KR20190003678A - Contols Body - Short Target Binding Agent - Google Patents

Abstract

The disclosure discloses a cyclic fusion polypeptide comprising a first portion of a binding domain, a second portion of a binding domain, and a spacer domain, wherein the spacer domain is a polypeptide and comprises at least 25 amino acid residues, Is a polypeptide and is fused to the N-terminus of the spacer domain through a first linker, the second portion of the binding domain is a polypeptide and is fused to the C-terminus of the spacer domain through a second linker, The second portion of the domain is associated with each other to form a binding site that specifically binds to the target.

Description

Contols Body - Short Target Binding Agent

The present invention belongs to the field of target binding molecules. The present disclosure reports a short chain binder comprising a spacer domain located between a first fragment of a binding domain and a second fragment of a binding domain.

Since Koehler and Milstein first developed monoclonal antibodies in 1974, much effort has been devoted to developing antibodies that are appropriate for human therapy. The first monoclonal antibodies available were developed in mice and rats. When used in human therapy, these antibodies are anti-rodent antibodies, resulting in unwanted side effects. Numerous efforts are being made to reduce or even eliminate these unwanted side effects.

Over the last several years, a large number of human monoclonal antibodies or humanized monoclonal antibodies have been on the market. Well known examples include, for example, Herceptin® and MabThera® from Hoffmann-La Roche (Basel).

In addition, novel antibody forms derived from wild-type, four chain, Y-type antibody forms have been developed. These forms are predominantly bispecific and multispecific. For review, see, for example, Kontermann, R., mAbs 4 (2012) 182-197.

US 2009/0175867 discloses a short chain polyvalent binding protein having an effector function.

WO 2014131711 reports bispecific antibodies in which the second and third antigen binding moieties can be fused directly or via the immunoglobulin hinge region to the Fc domain.

EP 15176083 discloses novel antibody forms and their use with reduced molecular weight as compared to full length antibodies.

WO 2007/048022 discloses antibody-polypeptide fusion proteins and methods of making and using the same.

WO 2007/146968 discloses short chain polyvalent binding proteins with effector functions.

EP 1 378 520 discloses cyclic single-stranded tri-specific antibodies.

Summary of the Invention

The present disclosure reports single cyclic polypeptides as novel target binders. In such cyclic polypeptides, the N-terminal portion comprises a first portion of the binding site and the C-terminal portion comprises a second portion of the binding site. The first portion of the binding site and the second portion of the binding site are associated with each other to form a complete or functional binding site. Thus, the polypeptide is cyclized. The meeting can be non-shared or shared. When the association is covalent, it is not by peptide bonding, but by, for example, a disulfide bond.

One aspect reported herein is a fusion polypeptide which (specifically) binds (cyclic) (short chain) to a target comprising a first portion of a binding domain, a second portion of a binding domain and a spacer domain, wherein

The spacer domain is a polypeptide (which forms a structural domain after folding)

- the first part of the binding domain is a polypeptide and is fused (directly or) through the first (peptide) linker to the N-terminus of the spacer domain,

- the second part of the binding domain is a polypeptide and is fused (directly or through the second (peptide) linker to the C-terminus of the spacer domain,

The first part of the binding domain (of the same (short chain) fusion polypeptide) and the second part of the binding domain (which are covalently or noncovalently associated with one another) and bind to the target specifically (binding) .

In one embodiment, the (annular) (short chain) fusion polypeptide comprises a precisely one portion of the N-terminal binding domain for the spacer domain and a different, precisely one portion of the same binding domain at the C-terminus for the spacer domain.

In one embodiment, the first portion of the binding domain is an antibody heavy chain variable domain and the second portion of the binding domain is an antibody light chain variable domain or vice versa.

In one embodiment, the first portion of the binding domain and the second portion of the binding domain are covalently associated with each other. In one embodiment, the first portion of the binding domain and the second portion of the binding domain are covalently associated with each other by a bond other than a peptide bond. In a preferred embodiment, the first portion of the binding domain and the second portion of the binding domain are covalently associated with each other by a disulfide bond.

In one embodiment, the first portion of the binding domain is an antibody heavy chain Fab fragment (VH + CHl) and the second portion of the binding domain is an antibody light chain Fab fragment (VL + CL) or vice versa.

In one embodiment, the binding site does not comprise a pair of antibody heavy chain variable domain and antibody light chain variable domain / antibody variable domain.

In a preferred embodiment, the spacer domain comprises at least 25 amino acid residues. In one embodiment, the spacer domain comprises at least 50 amino acid residues. In one embodiment, the spacer domain comprises at least 100 amino acid residues.

In one embodiment (annular) (short chain) fusion polypeptide exerts an effector function. In one embodiment, the effector function is ADCC and / or CDC.

In one embodiment, the spacer domain comprises an antibody hinge region or fragment thereof and an antibody CH2 domain or fragment thereof. In one embodiment, the hinge region and the antibody CH2 domain or fragment thereof are of the human IgGl subclass. In one embodiment, the hinge region and the antibody CH2 domain have the amino acid sequence of SEQ ID NO: 105.

In one embodiment, the spacer domain comprises an antibody hinge region or fragment thereof, an antibody CH2 domain, and an antibody CH3 domain or fragment thereof. In one embodiment, the hinge region, antibody CH2 domain, and antibody CH3 domain, or fragment thereof, are of the human IgGl subclass. In one embodiment, the hinge region, antibody CH2 domain, and antibody CH3 domain have an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-51. In one preferred embodiment, the hinge region, antibody CH2 domain, and antibody CH3 domain comprise the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 or SEQ ID NO: 38 or SEQ ID NO: 39 or SEQ ID NO: : ≪ / RTI > 41.

In one embodiment, the first and / or second linker is a peptide linker.

In one embodiment, the (cyclic) (short chain) fusion polypeptide that specifically binds to the target comprises a first portion of a binding domain, a second portion of a binding domain, and a spacer domain, wherein

The spacer domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, ,

The first portion of the binding domain is an antibody heavy chain Fab fragment and the second portion of the binding domain is an antibody light chain Fab fragment or vice versa,

-Binding domain is fused to the N-terminus of the spacer domain through the first peptide linker of SEQ ID NO: 64 or SEQ ID NO: 65 and the second portion of the binding domain is fused to the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: Terminus of the spacer domain through a second peptide linker of SEQ ID NO: 65, so that the first and second peptide linkers are selected independently of each other,

And

Antibody heavy chain Fab fragments and antibody light chain Fab fragments (associated with each other) form (functional) binding sites that (specifically) bind to the target.

In one embodiment, the (cyclic) (short chain) fusion polypeptide comprises exactly one antibody variable domain and spacer domain (i. E., A spacer domain) at the N-terminus to the C- Lt; / RTI > C-terminal to the spacer domain).

In one embodiment, the target is a soluble ligand of a cell surface antigen or a cell surface receptor.

In one embodiment, the binding domain is a conventional Fab, CrossFab or DutaFab.

In one embodiment, the binding domain is a conventional Fab, wherein a portion of the binding domain comprises at least the N-terminal fragment (or all) of the antibody heavy chain variable domain (VH) and the first antibody heavy chain constant domain (CHl) Each different binding domain comprises at least the N-terminal fragment (or all) of the antibody light chain variable domain (VL) and the antibody light chain constant domain (CL), or vice versa.

In one embodiment, a portion of the binding domain comprises VH-CHl in the C-terminal direction at the N-terminus and another portion of the binding domain comprises VL-CL in the C-terminal direction at the N- terminus, or vice versa to be.

In one embodiment, the binding domain is a CrossFab, wherein both parts of the binding domain comprise at least an N-terminal fragment (or all) of an antibody variable domain and an antibody constant domain, such that the pairs of variable domains and constant domains are natural Lt; / RTI > domain crossing / exchange of the heavy and light chain domains. In one embodiment, the exchange is the exchange of VH and VL or CH1 and CL.

In one embodiment, one portion of the binding domain comprises VL-CHl in the C-terminal direction at the N-terminus and the other portion of the binding domain comprises the VH-CL in the C-terminal direction at the N-terminus.

In one embodiment, a portion of the binding domain comprises VH-CL at the N-terminus in the C-terminal direction and another portion of the binding domain comprises VL-CHl in the C-terminal direction at the N-terminus.

The association of the (homologous) portion of the binding domain can be further facilitated by the introduction of charge compared to the domain exchange in CrossFab. In some cases (multicyclic) (dimeric cyclic) fusion polypeptides include at least a first (cyclic) fusion polypeptide and a second (cyclic) fusion polypeptide.

In one embodiment (multicyclic) fusion polypeptide is

a) a first (cyclic) fusion polypeptide comprising a Fab (specifically) binding to a first antigen as a binding site, and

b) a second (cyclic) fusion polypeptide comprising a Fab specifically binding to a second antigen as a binding site, wherein the variable domains VL and VH of the Fab (of the second circular fusion polypeptide) 2 (cyclic) fusion polypeptide

.

The (cyclic) fusion polypeptide in a) does not contain a modification as reported in b).

In the (cyclic) fusion polypeptide in b)

Within the antibody light chain fragment

The variable light chain domain VL is replaced by the variable heavy chain domain VH of the Fab,

Within the antibody heavy chain fragment

The variable heavy chain domain VH is replaced with the variable light chain domain VL of the Fab.

In one embodiment,

i) In the constant domain CL of the first (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide), the amino acid at a position corresponding to position 124 along Kabat is replaced by a positively charged amino acid, In the constant domain CH1 of the first (cyclic) fusion polypeptide of the polypeptide, the amino acid at the position corresponding to position 147 along the Kabat EU index or the position corresponding to position 213 along the Kabat EU index is a negatively charged amino acid Substituted,

or

ii) In the constant domain CL of the second (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide), the amino acid at the position corresponding to position 124 along Kabat is replaced by a positively charged amino acid, In the constant domain CH1 of the second (cyclic) fusion polypeptide, the amino acid at the position corresponding to position 147 along the kappa EU index or the position corresponding to position 213 along the kappa EU index is replaced with the negatively charged amino acid.

In one preferred embodiment,

i) an amino acid at position corresponding to position 124 according to Kabat in the constant domain CL of the first (cyclic) fusion polypeptide (of a multiple cyclic fusion polypeptide) is independently selected from the group consisting of lysine (K), arginine (R) or histidine (In a preferred embodiment, lysine (K) or arginine (R)), and in the constant domain CH1 of the first (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide) (E) or the aspartic acid (D), or the amino acid at the position corresponding to position 213 according to the kappa EU index is independently substituted with glutamic acid (E) or aspartic acid

or

ii) In the constant domain CL of the second (cyclic) fusion polypeptide (of a multiple cyclic fusion polypeptide), the amino acid at a position corresponding to position 124 along Kabat is independently selected from the group consisting of lysine (K), arginine (R) (Cyclic) fusion polypeptide of the (cyclic) fusion polypeptide of SEQ ID NO: 1 (in a preferred embodiment, lysine (K) or arginine (R) The amino acid at the position corresponding to 147 or the position corresponding to position 213 along the Kabat EU index is independently replaced by glutamic acid (E) or aspartic acid (D).

In one embodiment, in the constant domain CL of the second (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide), the amino acid at position 124 and 123 corresponding to the kappa EU index is replaced by K.

In the constant domain CH1 of the second (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide) in one embodiment, the amino acid at positions corresponding to positions 147 and 213 along the Kabat EU index is replaced by E.

In one embodiment, the binding domain is DutaFab, wherein a portion of the binding domain comprises at least the N-terminal fragment (or all) of the antibody heavy chain variable domain (VH) and the first antibody heavy chain constant domain (CHl) Domain comprises at least the N-terminal fragment (or all) of the antibody light chain variable domain (VL) and the antibody light chain constant domain (CL), wherein the binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain Wherein the first paratope comprises residues from CDR1 and CDR3 of the VL domain and from CDR2 of the VH domain and the second paratope comprises residues from the CDR1 and CDR3 and VL domains of the VH domain RTI ID = 0.0 > CDR2. ≪ / RTI >

One aspect reported herein is a (cyclic) fusion polypeptide comprising a first (cyclic) fusion polypeptide as reported herein and a second (cyclic) fusion polypeptide as reported herein, wherein the 1 and the second (cyclic) fusion polypeptide are the same or different, and the spacer domain of the first (cyclic) fusion polypeptide is (covalently) conjugated to the spacer domain of the second (cyclic) fusion polypeptide.

In one embodiment (double ring) fusion polypeptide is

A first (cyclic) (short chain) fusion polypeptide that (specifically) binds to a first target comprising a first portion of a binding domain, a second portion of a binding domain and a spacer domain, such as:

- the spacer domain has the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 38 or SEQ ID NO: 40,

The first portion of the binding domain is an antibody heavy chain Fab fragment and the second portion of the binding domain is an antibody light chain Fab fragment or vice versa,

-Binding domain is fused to the N-terminus of the spacer domain through the first peptide linker of SEQ ID NO: 64 or SEQ ID NO: 65 and the second portion of the binding domain is fused to the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: Terminus of the spacer domain through a second peptide linker of SEQ ID NO: 65, so that the first and second peptide linkers are selected independently of each other,

The antibody heavy chain Fab fragment and the antibody light chain Fab (fragment) (which are associated with each other) form a (functional) binding site that (specifically) binds to the first target,

And

A second (cyclic) (short chain) fusion polypeptide that (specifically) binds to a second target comprising a first portion of a binding domain, a second portion of a binding domain and a spacer domain, such as:

- the spacer domain has the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 39 or SEQ ID NO: 41,

The first portion of the binding domain is an antibody heavy chain Fab fragment and the second portion of the binding domain is an antibody light chain Fab fragment or vice versa,

-Binding domain is fused to the N-terminus of the domain through the first peptide linker of SEQ ID NO: 64 or SEQ ID NO: 65 and the second part of the binding domain is fused to the N-terminus of SEQ ID NO: 64 or SEQ ID NO: : 65 of the second peptide linker, so that the first and second peptide linkers are selected independently of each other,

The antibody heavy chain Fab fragment and the antibody light chain Fab fragment form a (functional) binding site that (specifically associates) with (binds to) the second target,

.

In one embodiment, each (cyclic) (short chain) fusion polypeptide comprises exactly one antibody variable domain and spacer domain (ie, a spacer domain) at the N-terminus to the spacer domain in the C- I.e., C-terminal to the spacer domain).

In one embodiment, the target is a soluble ligand of a cell surface antigen or a cell surface receptor.

In one embodiment, the binding domain is a conventional Fab, CrossFab or DutaFab.

In one embodiment, one or each of the binding domains is a conventional Fab, wherein a portion of the binding domain comprises at least the N-terminal fragment (or all) of the antibody heavy chain variable domain (VH) and the first antibody heavy chain constant domain (CHl) And each of the other binding domains comprises at least an N-terminal fragment (or all) of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL).

In one embodiment, a portion of the binding domain comprises VH-CHl in the C-terminal direction at the N-terminus and another portion of the binding domain comprises VL-CL in the C-terminal direction at the N- terminus, or vice versa to be.

In one embodiment, one or each of the binding domains is a CrossFab, wherein both parts of the binding domain comprise at least the N-terminal fragment (or all) of the antibody variable domain and antibody constant domain, and thus a pair of variable domains and constant domains Do not naturally associate with each other and have domain crossing / exchange of the heavy and light chain domains. In one embodiment, the exchange is the exchange of VH and VL or the exchange of CH1 and CL.

In one embodiment, one portion of the binding domain comprises VL-CHl in the C-terminal direction at the N-terminus and the other portion of the binding domain comprises the VH-CL in the C-terminal direction at the N-terminus.

In one embodiment, a portion of the binding domain comprises VH-CL at the N-terminus in the C-terminal direction and another portion of the binding domain comprises VL-CHl in the C-terminal direction at the N-terminus.

The association of the cognate binding domain can be further promoted by introducing the charge as compared to the domain exchange in the CrossFab. In this case, the (multicyclic) (dimeric cyclic) fusion polypeptide comprises at least a first (cyclic) fusion polypeptide and a second (cyclic) fusion polypeptide.

In one embodiment (multicyclic) fusion polypeptide is

a) a first (cyclic) fusion polypeptide comprising a Fab (specifically) binding to a first antigen as a binding site, and

b) a second (cyclic) fusion polypeptide comprising a Fab (specifically) binding to a second antigen as a binding site, wherein the variable domains VL and VH of a Fab (of the second (cyclic) fusion polypeptide) (Cyclic) fusion polypeptide < RTI ID = 0.0 >

.

The (cyclic) fusion polypeptide in a) does not contain a modification as reported in b).

In the (cyclic) fusion polypeptide in b)

Within the antibody light chain fragment

The variable light chain domain VL is replaced by the variable heavy chain domain VH of the Fab,

Within the antibody heavy chain fragment

The variable heavy chain domain VH is replaced by the variable light chain domain VL of the Fab.

In one embodiment,

i) In the constant domain CL of the first (cyclic) fusion polypeptide (of a multiple cyclic fusion polypeptide), the amino acid at a position corresponding to position 124 along Kabat is replaced by a positively charged amino acid, ) In the constant domain CH1 of the first (cyclic) fusion polypeptide, the amino acid at the position corresponding to position 147 along the Kabat EU index or the position corresponding to position 213 along the Kabat EU index is replaced by the negatively charged amino acid Or,

or

ii) In the constant domain CL of the second (cyclic) fusion polypeptide (of a multiple cyclic fusion polypeptide), the amino acid at a position corresponding to position 124 along Kabat is replaced by a positively charged amino acid, ) In the constant domain CH1 of the second (annular) fusion polypeptide, the amino acid at the position corresponding to position 147 along the Kabat EU index or the position corresponding to position 213 along the Kabat EU index is substituted with the negatively charged amino acid .

In one preferred embodiment

i) an amino acid at position corresponding to position 124 according to Kabat in the constant domain CL of the first (cyclic) fusion polypeptide (of a multiple cyclic fusion polypeptide) is independently selected from the group consisting of lysine (K), arginine (R) or histidine (In a preferred embodiment, lysine (K) or arginine (R)), and in the constant domain CH1 of the first (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide) (E) or the aspartic acid (D), or the amino acid at the position corresponding to position 213 according to the kappa EU index is independently substituted with glutamic acid (E) or aspartic acid

or

ii) In the constant domain CL of the second (cyclic) fusion polypeptide (of a multiple cyclic fusion polypeptide), the amino acid at a position corresponding to position 124 along Kabat is independently selected from the group consisting of lysine (K), arginine (R) (In a preferred embodiment, lysine (K) or arginine (R)), and in the constant domain CH1 of the second (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide) (E) or the aspartic acid (D) are independently substituted for the amino acid at the position corresponding to position 213 according to the kappa EU index.

In one embodiment, in the constant domain CL of the second (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide), the amino acid at position 124 and 123 corresponding to the kappa EU index is replaced by K.

In the constant domain CH1 of the second (cyclic) fusion polypeptide (of the multicyclic fusion polypeptide) in one embodiment, the amino acid at positions corresponding to positions 147 and 213 along the Kabat EU index is replaced by E.

In one embodiment, one or each of the binding domains is DutaFab, wherein a portion of the binding domain comprises at least the N-terminal fragment (or all) of the antibody heavy chain variable domain (VH) and the first antibody heavy chain constant domain (CHl) And each of the other binding domains comprises an antibody light chain variable domain (VL) and an N-terminal fragment (or all) of an antibody light chain constant domain (CL), wherein the binding domain comprises a heavy chain variable domain (VH) VL), wherein the first paratope comprises residues from CDR1 and CDR3 of the VL domain and from CDR2 of the VH domain and the second paratope comprises residues from the VH domain CDR1 and CDR3 and residues from CDR2 of the VL domain. One aspect reported herein is an isolated nucleic acid encoding a cyclic fusion polypeptide as reported herein.

One aspect reported herein is an isolated nucleic acid pair that codes together a dimeric (cyclic) fusion polypeptide as reported herein.

One aspect reported herein is a host cell comprising a nucleic acid as reported herein or a nucleic acid pair as reported herein.

One aspect reported herein is a method of producing a fusion polypeptide comprising culturing a host cell as reported herein to produce a (cyclic or double ring) fusion polypeptide and recovering the (cyclic or double ring) fusion polypeptide from the cell or culture medium (Cyclic or double ring) fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

One aspect reported herein is an immunoconjugate comprising a (cyclic) fusion polypeptide and a cytotoxic agent as reported herein.

One aspect reported herein is a pharmaceutical formulation comprising a (cyclic) fusion polypeptide as reported herein or a (dimeric) (cyclic) fusion polypeptide as reported herein and a pharmaceutically acceptable carrier .

One aspect reported herein is (cyclic) fusion polypeptides or dimeric (cyclic) fusion polypeptides as reported herein for use as pharmaceuticals.

One aspect reported herein is the use of (cyclic) fusion polypeptides as reported herein or dimeric (cyclic) fusion polypeptides as reported herein in the manufacture of medicaments.

DETAILED DESCRIPTION OF THE INVENTION

I. Definition

Knob into hole dimerization modules and their use in antibody manipulation are described in Carter P .; Ridgway J.B .; Presta L. G .: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73 (1).

General information on the nucleotide sequences of human immunoglobulin light and heavy chains can be found in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, .

As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are described in Kabat, et al., Seqeunces of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, 1991), and is referred to herein as " numbering according to Kabat ". In particular, the Kabat numbering system (see pages 647-660) of the Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, And the Kabat EU index numbering system (see pages 661-723) is used for the invariant heavy chain domain (in this case referred to herein as " numbering according to Kabat EU index " CH1, hinge, CH2, and CH3, which are more specific).

Methods and techniques useful in carrying out the present invention are described, for example, in the following references: [Ausubel, FM; (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N. D., and Hames, B. D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; [Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); [Watson, J. D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); [Winnacker, E. L., From Genes to Clones; N. Y., VCH Publishers (1987); [Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998)]; [Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).

The use of recombinant DNA technology enables the production of nucleic acid derivatives. Such derivatives can be modified by, for example, substitution, alteration, exchange, deletion or insertion, respectively, or positions of several nucleotide positions. Modification or derivatization can be performed, for example, through site directed mutagenesis. Such modifications are readily accomplished by those skilled in the art (see, for example, Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, BD, and Higgins, SG, Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

It should be noted that, in the present specification and the appended claims, the singular forms " a ", " a ", and " it " include plural references unless the context clearly dictates otherwise. Thus, for example, reference to " one cell " includes a number of such cells, and equivalents thereof known to those skilled in the art, and the like. Also, the terms " comprising ", " comprising ", " Can also be used interchangeably.

The term " about " means a range of +/- 20% of the numerical value subsequently followed. In one embodiment, the term " about " means a range of +/- 10% of the numerical value subsequently followed. In one embodiment, the term " about " means a range of +/- 5% of the numerical value that follows.

&Quot; Affinity " means the intensity of the sum of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the unique binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of the molecule X to its partner Y can generally be expressed as a dissociation constant (kd). The affinity can be measured in a general manner known in the art, including those described herein.

The term " amino acid sequence tag " refers to the sequence of amino acid residues linked to each other through peptide bonds with specific binding characteristics. In one embodiment, the amino acid sequence tag is an affinity or a purification tag. In one embodiment, the amino acid sequence tag is an Arg-tag, a His-tag, a Flag tag, a 3xFlag-tag, a Strep-tag, a Nano-tag, an SBP- tag, a c- Peptide, a cellulose-binding-domain, a chitin-binding-domain, a GST-tag, or an MBP-tag. In one embodiment, the amino acid sequence tag is SEQ ID NO: 01 (RRRRR), or SEQ ID NO: 02 (RRRRRR), or SEQ ID NO: 03 (HHHHHH), or SEQ ID NO: ID NO: 05 (DYKDDDDK) or SEQ ID NO: 06 (DYKDHDGDYK DHDIDYKDDD DK), or SEQ ID NO: 07 (AWRHPQFGG), or SEQ ID NO: 08 (WSHPQFEK) Or SEQ ID NO: 10 (MDVEAWLGAR VPLVET), or SEQ ID NO: 11 (MDEKTTGWRG GHVVEGLAGE LEQLRARLEH HPQGQREP), or SEQ ID NO: 12 (EQKLISEEDL), or SEQ ID NO: 13 (KETAAAKFER QHMDS) (Cell binding domain), or SEQ ID NO: 16 (cell binding domain), or SEQ ID NO: 17 (TNPGVSAWQV NTAYTAGQLV TYNGKTYKCL QPHTSLAGWE PSNVPALWQL Q), or SEQ ID NO: (GST-tag) or SEQ ID NO: 19 (MBP-tag).

The term " amino acid substitution " refers to the replacement of at least one amino acid residue of a predetermined parent amino acid sequence with a different " alternative " amino acid residue. Alternative residues or residues can be "naturally occurring amino acid residues" (ie, encoded by the genetic code) and are alanine (Ala); Arginine (Arg); Asparagine (Asn); Aspartic acid (Asp); Cysteine (Cys); Glutamine (Gln); Glutamic acid (Glu); Glycine (Gly); Histidine (His); Isoleucine (Ile): Leucine (Leu); Lysine (Lys); Methionine (Met); Phenylalanine (Phe); Proline (Pro); Serine; Threonine (Thr); Tryptophan (Trp); Tyrosine (Tyr); And valine (Val). In one embodiment, the replacement residue is not cysteine. Substitution by one or more non-naturally occurring amino acid residues is also encompassed by the definition of amino acid substitutions herein. By "non-naturally occurring amino acid residue" is meant a moiety other than the naturally occurring amino acid residues enumerated above that are capable of covalently bonding to the adjacent amino acid residue (s) within the polypeptide chain. Examples of unnatural amino acid residues include norleucine, ornithine, norvaline, homoserine, aib and other amino acid residue analogues such as Ellman, et al., Meth. Enzym. 202 (1991) 301-336. To generate such unnatural amino acid residues, the procedures of Noren et al. (Science 244 (1989) 182) and / or Ellman et al. (Homologous) can be used. Briefly, these procedures involve in vitro transcription and translation of RNA following chemical activation by non-naturally occurring amino acid residues of the suppressor tRNA. Non-naturally occurring amino acids can also be incorporated into peptides through chemical peptide synthesis and subsequent fusion of recombinantly prepared polypeptides, such as antibodies or antibody fragments, with these peptides.

The term " cytotoxicity of antibody-dependent cells (ADCC) " is a function mediated by Fc receptor binding and refers to the dissolution of target cells mediated by the antibody Fc-region in the presence of effector cells. The ADCC can be a multiple circular form as reported herein in the presence of effector cells such as freshly isolated PBMC (peripheral blood mononuclear cells) or effector cells purified from buffy coats such as monocytes or NK (natural killer) cells in one embodiment Is measured by treatment of a preparation of target expressing red blood cells (e.g., K562 cells expressing a recombinant target) into an Fc-region comprising a fusion polypeptide. The target cells are labeled with Cr-51 and then incubated with the multicyclic fusion polypeptide. The labeled cells are incubated with effector cells and the supernatant is analyzed for released Cr-51. Controls include the incubation of effector cells and target endothelial cells without multiple circular fusion polypeptides. The ability of the multicyclic fusion polypeptides to induce an early phase mediating ADCC is determined by measuring the binding of Fc [gamma] receptor-expressing cells, such as cells recombinantly expressing Fc [gamma] RI and / or Fc [gamma] RIA or NK cells (essentially expressing Fc [gamma] RIIIA) . In one preferred embodiment, the binding of Fc [gamma] R on NK cells is measured.

The term " association with " means binding of a binding site to its target, e.g., binding of an antibody binding region comprising an antibody heavy chain variable domain and an antibody light chain variable domain to an individual antigen. This association can be determined, for example, using a BIAcore® assay (GE Healthcare, Uppsala, Sweden).

For example, in one possible embodiment of the BIAcore (R) assay, the antigen is bound to the surface and the binding of the antibody binding site is measured by surface plasmon resonance (SPR). The affinity of the bond is defined by the term ka (binding constant: rate constant for binding forming the complex), kd (dissociation constant: rate constant for dissociation of the complex), and KD (kd / ka). Alternatively, the combined signal of the SPR sensorgram can be directly compared to the reference response signal for the resonance signal height and dissociation behavior.

The term " CH1 domain " means a portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 215 (EU numbering system). In one embodiment, the CH1 domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 20).

The term " CH2 domain " means a portion of an antibody heavy chain polypeptide extending from EU position 231 to EU position 340 (EU numbering system according to Kabat). In one embodiment, the CH2 domain has the amino acid sequence of APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 21). The CH2 domain is unique in that it does not pair closely with other domains. Instead, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native Fc-region. It has been speculated that carbohydrates may provide a substitute for domain-domain pairing and may help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206].

The term " CH3 domain " means a portion of an antibody heavy chain polypeptide extending from EU position 341 to EU position 446. In one embodiment, the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 22).

&Quot; Class " of an antibody or Fc-region refers to the type of constant domain or constant region that the heavy chain or fragment thereof retains. There are five main classes: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2 . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

As used herein, the term " cytotoxic agent " refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm- , Pb-212 and radioactive isotopes of Lu); Chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other inter An intercalating agent); Growth inhibitors; Enzymes and fragments thereof such as nucleolytic enzymes; Antibiotic; Toxins such as enzymatically active toxins or small molecule toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; And various antineoplastic or anti-cancer agents disclosed below.

The term " complement-dependent cytotoxicity " (CDC) refers to the lysis of cells induced by the Fc-region of an antibody as reported herein in the presence of complement. CDC is measured in a subject in the presence of a complement by treating the target expressing human endothelial cells with a polycyclic fused polypeptide as reported herein. The cells are labeled with calcein in one embodiment. CDC is identified when it induces lysis of 20% or more target cells at a concentration of 30 [mu] g / mL. Binding with the complement factor C1q can be measured by ELISA. In these assays, theoretically ELISA plates are coated with a multimeric cyclic fusion polypeptide in a concentration range, where purified human C1q or human serum is added. C1q binding is detected by an antibody directed against C1q, followed by a peroxidase-labeled conjugate. The detection of binding (maximum binding Bmax) was determined by measuring the optical density at 405 nm (OD405) against the peroxidase substrate ABTS® (2,2'-azino-di- [3- ethylbenzthiazoline-6-sulfonate] .

As used herein, the term " domain crossing " refers to a pair of antibody heavy chain VH-CHl fragment and its corresponding homologue antibody light chain, i.e., in an antibody binding arm (i.e., a Fab fragment), the domain sequence comprises at least one heavy chain domain Quot; means that the domain sequence is deviated from the natural sequence in that it is substituted by its corresponding light chain domain and vice versa. There are three common types of domain replacement: (i) a domain crossed heavy chain fragment (or a VH-CL-hinge-CH2-CH3 domain sequence with a VH-CL1 domain sequence and a domain crossed light chain and a VH- Have (Ii) a domain crossing light chain having a VH-CL domain sequence, and a domain crossing of a VH and VL domain causing a cross-domain heavy chain fragment having a VL-CHl domain sequence. (VL-CL) and a complete VH-CHl heavy chain fragment resulting in a domain crossed heavy chain fragment having a VH-CH1 domain sequence and a domain crossed heavy chain fragment having a VL-CL domain sequence (" Fab Cross-over ") (all the above-mentioned domain sequences are indicated in the C-terminal direction at the N-terminus).

As used herein, " substituted for each other " for the corresponding heavy and light chain domains means the above-mentioned domain crossing. Thus, if the CH1 and CL domains are " replaced ", this means the cross-domain mentioned under item (i) and the final heavy and light chain domain sequences. Thus, if VH and VL are " replaced ", this means the domain crossing mentioned under item (ii), and if CH1 and CL domains are replaced " This means the domain crossing mentioned under item (iii). Bispecific antibodies comprising domain crossings are described, for example, in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254 and Schaefer, W. et al, Proc. Natl. Acad. Sci USA 108 (2011) 11187-11192.

A multispecific antibody produced by a method as reported herein is essentially a crossover of the CH1 and CL domains as referred to under item (i) above, or a crossing of the VH and VL domains mentioned under item (ii) above Domain fragments. Fab fragments that specifically bind to the same antigen (s) are made to be of the same domain sequence. Thus, when more than one Fab fragment with a domain crossing is contained in a multispecific antibody, the Fab fragment (s) binds specifically to the same antigen.

&Quot; Effector function " refers to their biological activity due to the Fc-region of the antibody, which varies according to the antibody class from which it is derived. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; Antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytosis; Down regulation of cell surface receptors (e. G., B-cell receptors); And B-cell activation.

Fc receptor binding-dependent effector function may be mediated by the interaction of the Fc-region of the antibody with the Fc receptor (FcR), a specialized cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and through antibody-dependent cell mediated cytotoxicity (ADCC), the dissolution of a variety of other cellular targets (e. G., Tumor cells) presenting erythrocytes and Fc- Lt; RTI ID = 0.0 > (J) and < / RTI > ] Reference). FcR is defined as their specificity for immunoglobulin isotype: the Fc receptor for the IgG type Fc-region is referred to as Fc [gamma] R. Fc receptor binding is described, for example, in Ravetch, J.V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; [Capel, P. J., et al., Immunomethods 4 (1994) 25-34]; [de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; [Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of the receptor (Fc [gamma] R) to the Fc-region of an IgG-type antibody can be accomplished by a wide variety of methods including immunocomplex cleaning and control of antibody production, including phagocytosis, cytotoxicity of antibody- Triggers the effect function. In humans, three classes of Fc [gamma] Rs have been characterized and are as follows:

- FcγRI (CD64) binds to monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. At least one modification of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbered according to the EU index of Kabat) in Fc-region IgG reduces binding to Fc [gamma] RI. IgG2 residues at positions 233-236 substituted with IgG1 and IgG4 reduced binding to Fc [gamma] RI by 10 < 3 > times and eliminated the human monocyte response to antibody-sensitized red blood cells (Armor, KL, et al. Immunol. 29 (1999) 2613-2624).

- FcγRII (CD32) binds to complex IgG with medium to low affinity and is widely expressed. These receptors can be classified into two subtypes, Fc [gamma] RIA and Fc [gamma] RIIB. FcγRIIA is found on many cells involved in death (eg, macrophages, monocytes, neutrophils) and seems to be able to activate the death process. FcγRIIB appears to play a role in the inhibition process and is found on B cells, macrophages and mast cells and eosinophils. On B cells, this appears to function to inhibit further immunoglobulin production and, for example, isotype switching to the IgE class. In macrophages, Fc [gamma] RIIB acts as a mediator through Fc [gamma] RIA to inhibit phagocytosis. On eosinophils and mast cells, the B-form can help inhibit activation of these cells through IgE binding with its distinct receptors. The reduced binding to Fc [gamma] RIIA is detected in at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 Lt; RTI ID = 0.0 > Fc-region < / RTI > with mutations.

- FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two types. FcγRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on eosinophils. The reduced binding to Fc [gamma] RIIA can be achieved, for example, by the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 ≪ / RTI > numbering according to the EU index).

Mapping of binding sites on human IgG1 to Fc receptors, methods of measuring binding of the above-mentioned mutant sites and Fc [gamma] RI and Fc [gamma] RIAIA are described in Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604.

&Quot; Effective amount " of an agonist, e. G., A pharmaceutical formulation, refers to an amount effective, over a period of time and in a dose necessary to obtain a desired therapeutic or prophylactic result.

The term " epitope " refers to a portion of a given target required for a specific binding between a target and a binding site. The epitope can be continuous, that is, it can be formed by adjacent structural elements present in the target, or it can be discontinuous, i.e. it is a different position in the amino acid sequence of the protein as the primary sequence of the target, The three-dimensional structure employed in the environment, such as body fluids, is formed by closely related structural elements.

As used herein, the term "Fc receptor" refers to an activation receptor that is characterized by the presence of a cytoplasmic ITAM sequence associated with a receptor (see, eg, Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19 (2001) 275-290). These receptors include FcγRI, FcγRIIA, and FcγRIIIA. The term " unconjugated with Fc [gamma] R " means that the binding of the antibody as reported herein to NK cells at an antibody concentration of 10 [mu] g / mL is inhibited by binding to the anti-OX40L antibody LCOOl as reported in WO 2006/029879 Or 10% or less of the total weight of the sample.

Although IgG4 shows reduced FcR binding, antibodies from other IgG subclasses exhibit strong binding. However, when the pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., et al., J. Biol. Chem. 276 (2001) 6591-6604; Immunology 86 (1995) 319-324; and EP 0 307 434).

In one embodiment, the spacer domain of the cyclic short chain fusion polypeptide as reported herein is an antibody Fc-region.

In one embodiment, the Fc-region in a cyclic fusion polypeptide as reported herein is of the IgG1 or IgG2 subclass and includes mutant PVA236, GLPSS331, and / or L234A / L235A / P329G.

In one embodiment, the Fc-region in a cyclic fusion polypeptide as reported herein is of the IgGl subclass and comprises the mutation L234A / L235A. In one embodiment, the Fc-region in the annular fusion polypeptide further comprises the mutant P329G. In one embodiment, the Fc-region in a cyclic fusion polypeptide as reported herein is of the IgGl subclass and includes the mutant L234A / L235A / P329G (all numbered according to Kabat's EU index).

In one embodiment, the Fc-region in a cyclic fusion polypeptide as reported herein is of the IgG4 subclass and comprises the mutation L235E. In one embodiment, the Fc-region in the cyclic fusion polypeptide further comprises the mutation S228P. In one embodiment, the Fc-region in the annular fusion polypeptide further comprises the mutant P329G. In one embodiment, the Fc-region in a cyclic fusion polypeptide as reported herein is of the IgG4 subclass and includes the mutant S228P / L235E / P329G (all numbered according to Kabat's EU index).

As used herein, the term " Fc-region " is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term encompasses native sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226, or Pro230, or Ala231 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present.

A cyclic fusion polypeptide as reported herein may comprise an Fc-region, in one embodiment, an Fc-region derived from human origin. In one embodiment, the Fc-region comprises all portions of the human constant region. The Fc-region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. Binding to C1q is caused by binding sites defined within the Fc-region. Such binding sites are known in the art and are described, for example, in Lukas, TJ, et al., J. Immunol. 127 (1981) 2555-2560; [Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979) 907-917; [Burton, D. R., et al., Nature 288 (1980) 338-344); [Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004; [Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184; [Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; [Morgan, A., et al., Immunology 86 (1995) 319-324]; And EP 0 307 434. These binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to Kabat's EU index). The antibodies of the subclasses IgG1, IgG2 and IgG3 generally show complement activation, C1q binding and C3 activation, whereas IgG4 does not activate complement system, does not bind C1q, and does not activate C3. &Quot; Fc-region of an antibody " is a term well known to those skilled in the art and is defined based on the papain cleavage of the antibody. In one embodiment, the Fc-region is a human Fc-region. In one embodiment, the Fc-region is of a human IgG4 subclass comprising the mutations S228P and / or L235E and / or P329G (numbered according to the EU index of Kabat). In one embodiment, the Fc-region is of the human IgGl subclass comprising the mutations L234A and L235A and optionally P329G (numbered according to Kapp's EU index).

The term " hinge region " refers to a heavy chain variable region which, in the wild type antibody heavy chain, binds CH1 domain and CH2 domain, for example from about position 216 to about position 230 according to the EU numbering system of Kabat, Quot; refers to a portion of an antibody heavy chain polypeptide from about position 230 to about position 230. The hinge region of another IgG subclass can be determined by alignment with the hinge-region cysteine residue of the IgGl subclass sequence.

The hinge region is usually a dimer molecule composed of two polypeptides having the same amino acid sequence. The hinge region typically contains about 25 amino acid residues and is flexible to allow the associated target binding sites to move independently. The hinge region can be subdivided into three domains, upper, middle, and lower hinge domains (see, for example, Roux, et al., J. Immunol. 161 (1998) 4083).

In one embodiment, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 23), wherein X is S or P. In one embodiment, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 24), wherein X is S or P. In one embodiment, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 25), wherein X is S or P.

The term " wild-type Fc-region " means the amino acid sequence identical to the amino acid sequence of the Fc-region present in nature. The wild-type human Fc-region comprises natural human IgG1 Fc-regions (non-A and A allotypes), native human IgG2 Fc-region, native human IgG3 Fc-region, and native human IgG4 Fc- . The wild-type Fc-region comprises the amino acid sequence of SEQ ID NO: 26 (IgG1, a cocacyan allotype), SEQ ID NO: 27 (IgG1, African American Allotype), SEQ ID NO: 28 (IgG2) And SEQ ID NO: 30 (IgG4).

The term "variant (human) Fc-region" refers to an amino acid sequence that differs from the "wild-type" (human) Fc-region amino acid sequence due to at least one "amino acid mutation". In one embodiment, the variant Fc-region comprises at least one amino acid mutation, for example from about one to about ten amino acid mutations, and in one embodiment, from about one to about two amino acid mutations in the native Fc- It has five amino acid mutations. In one embodiment (variant) the Fc-region has at least about 80% homology with the wild-type Fc-region, and in one embodiment the variant Fc-region has at least about 90% homology, and in one embodiment the variant Fc- The region has at least about 95% homology.

Mutant (human) Fc-region is defined by the amino acid mutations that it contains. Thus, for example, the term P329G refers to a variant Fc-region having a proline glycine mutation at amino acid position 329 relative to the parent (wild type) Fc-region (numbered according to Kapp's EU index). The identity of the wild-type amino acid may not be specified, in which case the above-mentioned mutant is designated as 329G. The term "mutation" refers to a change to a non-naturally occurring amino acid, including a change to a naturally occurring amino acid (see, for example, US 6,586,207, WO 98/48032, WO 03/073238, US 2004/0214988, WO 2005/35727 , Chun, JW and Schultz, PG, Chem. Biochem. 11 (2002) 1135-1137 (2002), WO 2005/74524, Chin, JW, et al., J. Am. [Wang, L. and Schultz, PG, Chem. (2002) 1-10]); [Chin, JW, et al., PICAS United States of America 99 (2002) 11020-11024].

The polypeptide chain of the wild-type human Fc-region of the IgGl subclass has the following amino acid sequence beginning at the cysteine residue at position 227 and terminating at the glycine residue at position 446:

Variants of the IgGl subclass with the mutations T366S, L368A and Y407V The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variant of the IgGl subclass with mutant T366W The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A and L235A The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A, T366S, L368A and Y407V The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A and T366W The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A and P329G The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A, P329G, T366S, L368A and Y407V The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A, P329G and T366W The polypeptide chains of the human Fc-region have the following amino acid sequences:

Variants of the IgGl subclass with mutations L234A, L235A, P329G, Y349C, T366S, L368A and Y407V The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A, P329G, S354C and T366W The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A, P329G, S354C, T366S, L368A and Y407V The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations L234A, L235A, P329G, Y349C and T366W The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations I253A, H310A and H435A The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with the mutations H310A, H433A and Y436A The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgGl subclass with mutations M252Y, S254T and T256E The polypeptide chains of the human Fc-region have the following amino acid sequences:

The polypeptide chain of the wild-type human Fc-region of the IgG4 subclass has the following amino acid sequence:

Variants of the IgG4 subclass with mutations S228P and L235E The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgG4 subclass with mutations S228P, L235E and P329G The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgG4 subclass with mutations S228P, L235E, P329G, T366S, L368A and Y407V The polypeptide chain of the human Fc-region has the following amino acid sequence:

Variants of the IgG4 subclass with mutations S228P, L235E, P329G and T366W The polypeptide chain of the human Fc-region has the following amino acid sequence:

&Quot; Framework " or " FR " refers to variable domain residues other than hypervariable region (HVR) residues. FRs of a variable domain generally consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the VH (or VL) sequence as FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

The terms " full length antibody ", " intact antibody ", and " whole antibody " are used interchangeably herein to refer to antibodies having a structure substantially similar to a native antibody construct.

The terms " host cell, " " host cell line, " and " host cell culture " are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including offspring of such cells. Host cells include " transformants " and " transformed cells " that include progeny derived therefrom irrespective of transfected cells and passage number. The offspring may not be completely identical to the parent cell and the nucleic acid content, and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in native transformed cells are included herein.

&Quot; Humanized " antibody refers to a chimeric antibody comprising an amino acid residue derived from a non-human HVR and an amino acid residue derived from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVR (e. G., CDRs) , The whole or substantially all of the FR corresponds to that of a human antibody. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from the human antibody. &Quot; Humanized form " of an antibody, e. G., A non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term " hypervariable region " or " HVR " means that the sequence is hypervariable ("complementarity determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / , And an antigen-contacting residue (" antigen-contacting portion "). Generally, the antibody contains six HVRs, three of which are in VH (H1, H2, H3) and three of which are in VL (L1, L2, L3).

HVR

(a) is present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 A hypervariable loop (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917);

(b) the amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 CDR (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

(c) amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 Antigen contacts (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); And

(d) amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (A), (b), and / or (c) comprising (H2), 93-102 (H3), and 94-102

.

Unless otherwise indicated, the HVR residues and other residues (e.g., FR residues) of the variable domain are numbered herein in accordance with Kabat et al. (Homology).

An " immunoconjugate " is a cyclic fusion polypeptide as reported herein conjugated to one or more molecule (s) comprising a cytotoxic agent, without limitation.

An " individual " or " subject " is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., primates other than humans and humans such as monkeys), rabbits, and rodents And rats). In certain embodiments, the entity or object is a human.

An " isolated " cyclic fusion polypeptide, i. E. A double cyclic fusion polypeptide or a multicyclic fusion polypeptide, is isolated from its natural environment. In some embodiments, the cyclic fusion polypeptide is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) Gt; 95% < / RTI > or 99%. For a review of the evaluation of purity, see, for example, Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

&Quot; Isolated " nucleic acid refers to a nucleic acid molecule that is isolated from a component of its natural environment. The isolated nucleic acid usually comprises nucleic acid molecules contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present at a chromosomal site other than or at a different chromosomal location than its natural chromosomal location.

An " isolated nucleic acid encoding a (poly) annular fusion polypeptide " refers to an isolated nucleic acid molecule comprising a nucleic acid molecule (s) in a single vector or discrete vector, and such nucleic acid molecule (s) Quot; means a nucleic acid molecule of one (homologous multimeric (multiple) cyclic fusion polypeptide) or more (heteromultimeric (multiple) cyclic fusion polypeptide) that each encode a short chain polypeptide of the polypeptide.

The term " light chain " means a shorter polypeptide chain of a native IgG antibody. The light chain of an antibody may be assigned to one of two types, called kappa (lambda) and lambda (lambda), based on the amino acid sequence of its constant domain, and may be designated as SEQ ID NO: 52 for the human kappa light chain constant domain and human lambda light chain See SEQ ID NO: 53 for the constant domain.

As used herein, the term " monoclonal antibody " refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population bind identical and / or identical epitopes, Or variant antibodies raised during the preparation of monoclonal antibody preparations are exceptional, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single crystal moiety on the antigen. Thus, the qualifier " monoclonal " refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the manufacture of antibodies by any particular method. For example, monoclonal antibodies to be used in accordance with the invention include, but are not limited to, hybridoma methods, recombinant DNA methods, phage-display methods, and methods employing transgenic animals containing all or part of a human immunoglobulin locus Can be produced by a variety of techniques, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

&Quot; Naked annular fusion polypeptide " means a cyclic fusion polypeptide that is not conjugated to a moiety (e. G., A cytotoxic moiety) or a radioactive label. Bile annular fusion polypeptides can be present in pharmaceutical formulations.

&Quot; Natural antibody " means a naturally occurring immunoglobulin molecule having a variety of structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light chains and two identical heavy chains disulfide-linked. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain or heavy chain variable domain, and subsequently three constant domains (CH1, CH2, and CH3) The hinge region is located between the two constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain or light chain variable domain, and subsequently an unchanging light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (lambda) and lambda (lambda), based on the amino acid sequence of its constant domain.

The term " package insert " is used to refer to an instruction that is normally included in a commercial package of a therapeutic product, including instructions, usage, dosage, administration, combination therapy, contraindications and / As shown in FIG.

The term " paratope " refers to the portion of a given antibody molecule that is required for specific binding between a target and a binding site. The paratope may be continuous, i.e., formed by adjacent amino acid residues present at the binding site, or may be discontinuous, i.e., as in the amino acid sequence of the CDR of the amino acid residue, at a different position in the primary sequence of the amino acid residue , But the binding site can be formed by amino acid residues that are closely related in the tertiary structure employed.

The term " peptide linker " means a linker of natural and / or synthetic origin. Peptide linkers consist of a linear chain of amino acids, in which the 20 naturally occurring amino acids are monomer building blocks linked by peptide bonds. The chain has a length of from 1 to 50 amino acid residues, preferably from 1 to 28 amino acid residues, particularly preferably from 3 to 25 amino acid residues. The peptide linker may contain a naturally occurring polypeptide sequence or a repetitive amino acid sequence. Peptide linkers have the function of ensuring that the domains of the annular fusion polypeptide can perform their biological activity by allowing these domains to fold properly and to be appropriately present. Preferably, the peptide linker is a " synthetic peptide linker " designated to be rich in glycine, glutamine, and / or serine residues. These linkers include, for example, small repeat units of up to five amino acids, such as GGGS (SEQ ID NO: 54), GGGGS (SEQ ID NO: 55), QQQG (SEQ ID NO: 56), QQQQG 57), SSSG (SEQ ID NO: 58) or SSSSG (SEQ ID NO: 59). (GGGS) 3 (SEQ ID NO: 61), (GGGS) 4 (SEQ ID NO: 62), (GGGS) (SEQ ID NO: 63), (GGGGS) 2 (SEQ ID NO: 64), (GGGGS) 3 To 5 times. At the amino-terminal and / or carboxy-terminal of the multimer, up to six additional optional, naturally occurring amino acids may be added. Other synthetic peptide linkers consist of a single amino acid that is repeated 10 to 20 times, such as, for example, a serine at the linker GSSSSSSSSSSSSSSSG (SEQ ID NO: 67), and at the amino-terminal and / or carboxy- And may include any naturally occurring amino acid. All peptide linkers can be encoded by nucleic acid molecules and thus recombinantly expressed. Since the linker is itself a peptide, an anti-fusogenic peptide is linked to the linker by a peptide bond formed between the two amino acids.

&Quot; Amino acid sequence identity percentage (%) " for a reference polypeptide sequence refers to the percentage identity of the amino acid sequence identity of the reference polypeptide sequence to that of the reference polypeptide sequence after aligning the sequence and introducing a gap to obtain a percentage of maximum sequence identity if necessary, Is defined as the percentage of amino acid residues of the same candidate sequence as the amino acid residues of the reference polypeptide sequence. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways pertaining to the art using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) ≪ / RTI > One of ordinary skill in the art can determine any suitable parameters for aligning the sequence, including any algorithms needed to obtain maximum alignment over the entire length of the sequence being compared. However, for purposes herein,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code was submitted with the user documentation to the U.S. Copyright Office (Washington D.C., 20559), where it was registered with US copyright registration number TXU510087. The ALIGN-2 program was developed by Genentech, Inc. (South San Francisco, Calif.), Or may be compiled from source code. The ALIGN-2 program must be compiled for use on UNIX operating systems that include Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not variable.

In the situation where ALIGN-2 is applied to the amino acid sequence comparison, for a given amino acid sequence B, the amino acid sequence identity% of the given amino acid sequence A against it, or against it (alternatively for a given amino acid sequence B, Or may be expressed as a predetermined amino acid sequence A that has or comprises a constant amino acid sequence identity% against it) is calculated as follows:

100 x fraction X / Y

Where X is the number of amino acid residues scored as the same match by the sequence alignment program ALIGN-2 in the alignment of the program's A and B and Y is the total number of amino acid residues of B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless otherwise stated, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term " pharmaceutical formulation " means a formulation that is effective in the biological activity of the active ingredient contained therein and does not contain any additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

&Quot; Pharmaceutically acceptable carrier " means, in addition to the active ingredient, a component in the pharmaceutical formulation which is harmless to the subject. Pharmaceutically acceptable carriers include, without limitation, buffers, excipients, stabilizers, or preservatives.

As used herein, " treatment " (and grammatical variations thereof such as "treating" or "treating") refers to a clinical intervention attempting to alter the natural course of the subject being treated, Can be performed during the process. The beneficial effects of the treatment include, without limitation, prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of disease progression, Includes roadway or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of the disease or delay the progression of the disease.

The term " variable region " or " variable domain " means the domain of an antibody heavy chain or light chain that participates in the binding of an antigen to an antibody. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR) See, for example, Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domains from an antibody that binds to the antigen, respectively, to screen libraries of complementary VL or VH domains (see, e.g., Portolano, S. et al. , J. Immunol. 150 (1993) 880-887; Clackson, T., et al., Nature 352 (1991) 624-628).

As used herein, the term " vector " means a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid construct, as well as a vector integrated into the genome of the host cell introduced therein. Constant vectors can induce the expression of nucleic acids to which they are operatively linked. Such a vector is referred to herein as an " expression vector ".

The invention is illustrated below using cyclic fusion polypeptides of different structure and specificity. They are presented only to illustrate the invention. This should not be construed as a limitation. The true category is described in the claims.

II. The cyclic fusion polypeptides reported herein

The present invention is based, at least in part, on the discovery that the fusion of light chain variable domains to the C-terminus of the (full length) heavy chain, or vice versa, results in the formation of functional binding sites for the respective VH and VL domains That is, a pair of variable light and variable heavy chain domains within a single polypeptide chain forms functional VH / VL-pair to form a functional binding site by intramolecular cyclization.

The present invention is based, at least in part, on the discovery that a target binding agent comprising a single (cyclic) polypeptide can be provided. In such (circular) polypeptides, the N-terminal portion comprises a first portion of a binding domain and the C-terminal portion comprises a second portion of a binding domain. The first portion of the binding domain and the second portion of the binding domain (associate with each other) form a complete or functional binding site. Thus, the polypeptide is cyclized.

The present invention is based, at least in part, on the ability of the final (double ring) dimer fusion < RTI ID = 0.0 > (Fc) < / RTI > fusion to be achieved by modifying the length of a peptide linker linking individual antibody variable domains to separate N- and C- It is based on the discovery that it is possible to adjust the geometry / distance of two binding sites in a polypeptide.

The present invention is based, at least in part, on the discovery that the binding geometry of a dimer, i.e., double ring, fusion polypeptide, can vary according to the length of the first and second linkers (i. E., The linker length ratio). It is thus possible to fix the geometry of the two Fab-like coupling arms to each other.

Disclosed herein are cyclic fusion polypeptides comprising a first portion of a binding domain, a second portion of a binding domain, and a spacer domain, wherein

- the spacer domain is, for example, a polypeptide that forms a structural domain after folding,

- the first part of the binding domain is a polypeptide and is fused to the N-terminus of the spacer domain through a first (peptide) linker,

The second portion of the binding domain is a polypeptide and is fused to the C-terminus of the spacer domain through a second (peptide) linker,

The first portion of the binding domain (of the same fusion polypeptide) and the second portion of the binding domain are associated with each other to form a binding site that specifically binds to the target (functional).

The first portion of the binding domain and the second portion of the binding domain may be associated non-covalently or covalently with each other. If the association is covalent, it is due to a bond other than a peptide bond, for example a disulfide bond.

The spacer domain is a polypeptide that forms a structural domain after folding. Thus, the spacer domain may be smaller than the 100 amino acid residues, but needs to be structurally restricted to immobilize the binding motif. Exemplary spacer domains are a dimer coil-coil, an antibody hinge region, or an antibody Fc-region or fragment thereof.

The cyclic fusion polypeptides reported herein are short chain polypeptides. The general structure of the cyclic fusion polypeptide is shown in Fig.

The disclosure also discloses dimers, i.e., double ring, fusion polypeptides, comprising a first cyclic fusion polypeptide as reported herein and a second cyclic fusion polypeptide as reported herein, wherein the first and second circular fusion polypeptides, The bicyclic fusion polypeptide is the same or different and the spacer domain of the first annular fusion polypeptide is conjugated to the spacer domain of the second annular fusion polypeptide by at least one non-peptide bond, in one embodiment at least one disulfide bond, do.

In this case, the spacer domain is a dimerization domain, i.e. the structural property of the spacer domain is the provision of dimerization functionality.

Similarly, trimers, i.e., triple rings, fusion polypeptides and tetramers, i.e., quadruple, fusion polypeptides, can be obtained when the trimerization domain or tetramerization domain is used as the spacer domain, respectively.

Polycyclic fusion polypeptides as reported herein are also referred to as Contorsbodies. The general structure of the double ring fusion polypeptide / contol body is shown in Figure 2a (with dimerization spacer domain) and 2b (without dimerization spacer domain) and the general structure of the triple ring fusion polypeptide / , And the general structure of the quadruple fusion polypeptide / contol body is shown in Figure 4a (with tetramer spacer domain) and 4b (without tetramer spacer domain).

If the spacer domain is an antibody Fc-region, as an example of a dimerization domain, and the binding domain is an antibody Fab heavy chain and light chain fragment, then this specific contoll body is in antibody form. Compared to normal IgG antibodies, the contoll body utilizes only one chain and is not a heavy chain or light chain. The unique feature of the contexts body is that the Fc-region coding sequence is located between the heavy chain Fab fragment and the light chain Fab fragment.

These specific contexts bodies are used below to demonstrate the specific properties of the double-ring fusion polypeptides as reported herein. In addition to the Fab fragment, an isolated variable domain may also be used as part of the binding site.

Short chain fusion polypeptides as reported herein having a "hinge-CH2-CH3" spacer domain as the dimerization domain between the heavy and light chain Fab fragments are dimerized through their Fc-region, ≪ / RTI > where the two Fabs are fixed in orientation relative to each other. In contrast, the two Fabs in the normal IgG type antibody are more flexible and oriented much differently.

The geometry of the two joining sites to each other can be adjusted in the context body by the length of the linker to which it is applied. The alignment and spatial distance between the binding sites of the double-ring fusion polypeptides and the IgG type of normal antibodies as reported herein in the exemplary embodiments are shown in FIG.

The spatial distance between binding sites for conventional antibodies of the IgG type is about 80 Å or more. The spatial distance between the binding sites of the double-ring fusion polypeptides as reported herein can be from about 20 to about 50, depending on the length and length ratio of the peptide linker in the individual cyclic fusion polypeptides that form the double-ring fusion polypeptide . Depending on the intended geometry, a peptide linker is selected. In one embodiment, the peptide linker is independently selected from the group of peptide linkers consisting of SEQ ID NO: 54 to SEQ ID NO: 70.

II.1. The single specific multinucleated fusion polypeptides reported herein

Monospecific polycyclic fused polypeptides include two or more cyclic fused polypeptides as reported herein wherein each cyclic fused polypeptide specifically binds to the same epitope on the same target, i. E., Comprises the same binding site .

Exemplary monospecific multiresponse fusion polypeptides are anti-Her2 contracts bodies (including cyclic fusion polypeptides of SEQ ID NO: 96). This was prepared by transfecting HEK293 cells with a vector containing a nucleotide sequence encoding a circular fusion polypeptide.

It has been found that conventional protein A affinity chromatography is suitable for extracting the Fc-region containing portion of the culture supernatant. Alternatively, a hexa histidine C-terminal tag (SEQ ID NO: 03) linked via a GSG peptide linker can be used for purification.

Preparative size exclusion chromatography was used in the second purification step to isolate cyclic fusion polypeptides from product-related impurities, which are usually high-dimensional structures of the cyclic fusion polypeptide (small aggregates were also identified in the chromatogram as in the case of IgG type antibodies ) (For example, see Fig. 15). Typical yields of these constructs averaged 10 mg / liter. An anti-Her2 contexts body was expressed in several batches (0.5 liter to 2 liter shake flask scale). The product quality was analyzed by mass spectrometry (see FIG. 6). The identity of the product was confirmed with a purity rating of greater than 95%.

The binding of the anti-Her2 context body in its double-ringed form (see Figure 2a) and its quadruple form (see Figure 4b) assesses the affinity and the synthesis of the molecule as compared to the conventional anti-Her2 antibody of the IgG type (SPR, e. G., BIAcore) in two different settings to determine < / RTI >

In the first setting (in the case of 1: affinity in the table below), each anti-Her2 context body was captured with an anti-human Fc-region antibody conjugated to the surface of the SPR chip. The Her2 extracellular domain (ECD) was used as the analyte. In the second setting (2 in the table below, in the case of the synthesis), the anti-Her2 antibody putuzumab (sold as Perjeta (R)) was immobilized on the chip surface and thus captured the ECD of Her2. Each con- tent body was used as the analyte. The synthesis of the IgG type reference antibody trastuzumab, bivalent anti-Her2 contexts bodies and tetravalent anti-Her2 contexts bodies was measured using a series of concentrations of the analyte. The anti-Her2 antibody trastuzumab (sold as Herceptin (R)) was used as a basis for both settings.

The Contes body is much denser compared to conventional antibodies of the IgG type.

The anti-Her2 context body was tested in the proliferative assay. Similar to Scheer et al. (Scheer et al., PLoS One 7 (2012) e51817) using a chemically cross-linked trastuzumab Fab, the anti-Her2 con- ttus body mobilized receptors on the cell surface and promoted activation signals . Trastuzumab, a binding agent for the Her2 epitope located on the receptor stem base domain, keeps the receptors away from each other and consequently antagonizes proliferation. Figure 7 shows the differential effect of the anti-proliferative and proliferative, individual Trastuzumab and double-ring and quadruple anti-Her2 contexts bodies.

The ability of the anti-Her2 context body to bind to the FcRn receptor was determined. The binding to both the human FcRn receptors of both anti-Her2 con- ttus bodies, as compared to the IgG type, an antibody of the IgG1 subclass, was surprisingly higher, that is, the binding to both the cyclic and the quadruple con- tent bodies (See Fig. 8A). Condoms bodies behaved similarly to cynomolgus FcRn trastuzumab (see Fig. 8b), the dimeric con- tors body was 4.5 times more affine than trastuzumab and the tetrameric con- ts body was 61 times more affine .

The ability of anti-Her2 context bodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated. Through binding with Fc [gamma] R, IgG can trigger ADCC. In Figure 9, the ADCC kinetics of the anti-Her2 con- tents body and trastuzumab are shown.

Utilizing tryptophan autofluorescence, the first thermal denaturation temperature T _m 1 is approximately 63 ° C relative to the contus body. Using static light scattering, an aggregation initiation temperature of approximately 66 占 폚 was determined for the Constant body. Using dynamic light scattering, a flocculation onset temperature of approximately 66 占 폚 was determined for the Constant body. In all experiments the formulation was 20 mg His / His * HCl, 1 mg / mL Kontels body in 140 mM NaCl.

It was confirmed by mass spectrometry that no mixed Fab was formed.

Other examples of double-ringed monospecific contont bodies include anti-CD1cont bodies (SEQ ID NO: 97), and anti-CD20 con- ttus bodies with different variable domains ((1) SEQ ID NO: (2) a cyclic fused polypeptide of SEQ ID NO: 99). In the table below, the expression rates, yield and quality of these contoll bodies are provided.

All the contoll bodies were transiently expressed in HEK-293 cells at a yield ranging from 2 to 15 mg / L. The product after the protein A column was more than 85% and was purified from byproducts to a purity of generally over 96% by SEC column chromatography.

II.2. A multispecific multiresponse fusion polypeptide

A multispecific multiresponse fusion polypeptide comprises two or more annular fusion polypeptides as reported herein wherein each cyclic fusion polypeptide specifically binds to a different target and / or different epitope on the same target, i. E., Different specificity At least two of the bonding sites.

Not to be limited to this theory, the self-assembly of the corresponding domains of the binding site in the short chain polypeptide is more dominant than the interchain association, i.e., after expression of the monocyclic fusion polypeptide, the individual, non-functional domains of the binding site are associated, To form a binding site. For example, if the functional binding site is a Fab and the spacer domain is an Fc-region, the Fab moiety, the heavy chain fragment (VH-CH1) and the light chain fragment (VK-CK) , One half of the Fc-portion of the first assembled annular fusion polypeptide associates subsequently with the other cyclic fusion polypeptide to form a double ring fusion polypeptide (a contoll body comprising dimers of two monocyclic fusion polypeptides). The heterodimerization of isolated fusion polypeptides should be promoted to obtain multispecific multispecific fusion polypeptides. One exemplary heterodimerization promoting element is a mutation according to the knob-into-hole technique.

It is possible to modify the length of the linker at the N-terminus and C-terminus of each of the Fc-region spacer domains to change the geometry / distance of the two binding sites of the final bispecific contol body, The same is true of the Tols body.

It is possible to modify the geometry / distance of the joining site by changing the relative position (s) of the joining site (s) with respect to each other.

For example, in the case of a Fab as a binding site, the sequence of the domains of the heavy chain and light chain Fab fragments can be deduced, i.e., for example, the heavy chain Fab fragments comprise the domain sequence VH-CH1 or CH1-VH (N- Terminus to the C-terminus), and similarly the light chain Fab fragment may have the domain sequence VL-CL or CL-VL (at the N-terminus to the C-terminus) or may be mixed. Assuming that the linker is present before and after the spacer domain, for example before and after the Fc-region (in the C-terminal direction at the N-terminus), the Fab fragment is restricted to its orientation relative to the spacer domain. As a result, the relative positions of the VH domains become closer (or referred to herein as " VH-in ") or farther away from the Fc- 11).

It is also possible to modify the assembly behavior using CrossMab technology, i.e., domain swapping on one arm. This can further be combined with the charge variant in the exchange or non-exchange arm. Exemplary chains of the individual orientation anti-cMET cyclic fusion polypeptides used in the production of the bispecific, double-ring fusion polypeptides are shown in Figure 12 and show the individual expression rates, yields, VH-out, VH- And quality are provided in the following table.

VH-out-knob = SEQ ID NO: 100,

VH-in-knob = SEQ ID NO: 101,

VH-out-hole = SEQ ID NO: 102,

VH-out-hole-CH-CL-bridging = SEQ ID NO: 103,

VH-in-hole-VH-VL-bridging = SEQ ID NO: 104.

A cyclic fusion polypeptide as reported herein may comprise any type of two-component or multi-component complex, provided that the multimerization domain, e.g., the half Fc-region, can be inserted into the sequence of the final short chain polypeptide. An example of such a multi-component complex is a peptide-loaded MHC-I complex. The individual context bodies are shown in Fig.

The effects based on MHC-I mediated kill cell mobilization and cell clearance are similar between the bisulfite fusion polypeptides reported herein and the MHC-I IgG-type antibody fusions (Figure 14).

Techniques generally applicable to the production of conventional multispecific antibodies can also be used and adapted to produce multispecific multiresponse fusion polypeptides as reported herein.

For example, techniques for producing multispecific antibodies include, without limitation, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificity (Milstein, C. and Cuello, AC, Nature 305 (1983) (See, for example, U.S. Pat. No. 5,731,168), WO 93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) ). Multispecific antibodies may also be used to manipulate electrostatic steering effects for the production of antibody Fc-heterodimeric molecules (WO 2009/089004); Cross-linking of two or more antibodies or fragments (see, for example, US 4,676,980 and Brennan, M. et al. , Science 229 (1985) 81-83); The use of leucine zippers for the production of bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); (See, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448) for the preparation of bispecific antibody fragments ; And the use of short chain Fv (sFv) dimers (see, for example, Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); And for example, Tutt, A. et al., J. Immunol. 147 (1991) 60-69. ≪ / RTI >

In one embodiment of all aspects, the cyclic fusion polypeptide as reported herein is a multispecific multiple cyclic fusion polypeptide that requires heterodimerization of at least two cyclic fusion polypeptides.

Similarly, one aspect reported herein is a cyclic fusion polypeptide of a multimer, preferably a dimer, as reported herein, wherein the first cyclic fusion polypeptide specifically binds to a first target, The bicyclic fusion polypeptides specifically bind to a second target, each comprising a heterodimerization domain as a spacer domain.

The Fc-region contained in the contexts body may have FcR effector function (wild type; SEQ ID NO: 31) or absent (no Fc gamma III effector function with mutations L234A, L235A, P329G : No FcRn effector function with mutations I253A, H310A, H435A (SEQ ID NO: 44) or H310A, H433A, Y436A (SEQ ID NO: 45); numbered according to Kabat EU index).

Binding site includes an antibody binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain pair, an FcRn comprising an MHC-like domain and a beta-2-microglobulin, or a DARPINS (designated an anchin repeat domain), a tandem scFv , And a pair of two consecutive anticalines.

In general, whenever a geometric constraint is required to anchor the orientation of the two binding sites, a contus body may be used.

In one embodiment, the spacer domain comprises a tag. In one embodiment, the tag is conjugated to the C-terminus of the annular fusion polypeptide.

In one embodiment, the spacer domain comprises a multimerization domain. In one embodiment, the multimerization domain is selected from the group consisting of an antibody Fc-region and variants thereof, and tetranectin domains and variants thereof.

The spacer domain may be a single coil domain that forms a multimer, or it may be a multifunctional, native protein that is multimerized and is known to confer its own function to the contexts body, as a multimer assembly. For example, the Myc / Max / Mad family dimer or leucine zipper produces a dimer coil-coil, COMP (cartilage oligomeric matrix protein) has a head-to-head orientation and a dimer coil- Create a similar system. Several other systems with coil-coils with a disulfide bridge useful for having head-to-head orientation, dimerization domains, Fabs fixed at 180 degrees, such as SARAH (human MST1 C- terminal dimerization domain (residues 431-487) Lt; / RTI > Also, a tenacin-C (TNC) trimerization domain can be used.

III. Binding site

III.1. Antibody fragment-derived binding site

In certain embodiments, the binding site of a cyclic fusion polypeptide as reported herein consists of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL).

In certain embodiments, the binding site of the annular fusion polypeptide is an antibody fragment. Antibody fragments include, without limitation, Fab, Fab ', Fab'-SH, and Fv fragments. For a review of certain antibody fragments, see Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134. For a review of the scFv fragment, see, for example, Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315, and also WO 93/16185; See US 5,571,894 and US 5,587,458. For a review of Fab fragments containing a collection receptor binding epitope residues and increased in vivo half life, see US 5,869,046.

The antibody fragment may also be a " bi-functional Fab " or " DAF " (see, for example, US 2008/0069820).

Single-domain antibodies are antibody fragments that include all or part of the light chain variable domain of the antibody, or all or part of the heavy chain variable domain. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).

Antibody fragments can be prepared by a variety of techniques, including, without limitation, preparation of recombinant host cells (e. G. E. coli or phage), including the hydrolytic digestion of intact antibody proteins as described herein .

If the binding site is an Fab, the Fab can be a conventional Fab, CrossFab or DutaFab.

In the case of conventional Fab, a portion of the binding domain comprises at least the N-terminal fragment (or all) of the antibody heavy chain variable domain (VH) and the first antibody heavy chain constant domain (CHl), and each of the other binding domains comprises an antibody light chain At least the N-terminal fragment (or all) of the variable domain (VL) antibody light chain constant domain (CL). The order of these domains may be arbitrary if it is possible to associate them and to form (functional) binding sites (i.e., not interfere).

In one embodiment, a portion of the binding domain comprises VH-CHl in the C-terminal direction at the N-terminus and another portion of the binding domain comprises VL-CL in the C-terminal direction at the N-terminus.

In the case of CrossFab, both parts of the binding domain contain at least the N-terminal fragment (or all) of the antibody variable domain and antibody constant domain so that the pair of variable domains and constant domains do not naturally associate with each other, Domain cross / exchange. This may be the exchange of VH and VL or the exchange of CH1 and CL. The order of these domains may be arbitrary if it is possible to associate them and to form (functional) binding sites (i.e., not interfere).

In one embodiment, one portion of the binding domain comprises VL-CHl in the C-terminal direction at the N-terminus and the other portion of the binding domain comprises the VH-CL in the C-terminal direction at the N-terminus.

In one embodiment, a portion of the binding domain comprises VH-CL at the N-terminus in the C-terminal direction and another portion of the binding domain comprises VL-CHl in the C-terminal direction at the N-terminus.

In the case of multiple cyclic fusion polypeptides, association of a cognate binding domain can be further facilitated by the introduction of a charge as compared to domain exchange in CrossFab. Wherein the multicyclic fusion polypeptide comprises at least a first annular fusion polypeptide and a second annular fusion polypeptide.

In one embodiment, the multinucleated fusion polypeptide comprises

a) a first annular fusion polypeptide comprising a Fab specifically binding to a first antigen as a binding site, and

b) a second annular fusion polypeptide comprising a Fab specifically binding to a second antigen as a binding site, wherein the variable domains VL and VH of the Fab (of the second circular fusion polypeptide) are replaced by a second annulus Fusion polypeptide

.

The cyclic fusion polypeptide under a) does not contain a modification as reported under b).

b) For a circular fusion polypeptide underneath,

Within the antibody light chain fragment,

The variable light chain domain VL is replaced by the variable heavy chain domain VH of the Fab,

Within the antibody heavy chain fragment,

The variable heavy chain domain VH is replaced with the variable light chain domain VL of the Fab.

In one embodiment,

i) In the constant domain CL of the first annular fusion polypeptide of the multicyclic fusion polypeptide, the amino acid at a position corresponding to position 124 along Kabat is replaced by a positively charged amino acid and the first annular fusion polypeptide of the multicyclic fusion polypeptide In the constant domain CH1, the amino acid at the position corresponding to position 147 according to the Kabat EU index or the amino acid at the position corresponding to position 213 according to the Kabat EU index is substituted with a negatively charged amino acid,

or

ii) In the constant domain CL of the second annular fusion polypeptide of the multiple cyclic fusion polypeptide, the amino acid at a position corresponding to position 124 along the Kabat is replaced by a positively charged amino acid and the second cyclic fusion polypeptide of the multiple cyclic fusion polypeptide In the constant domain CH1, the amino acid at the position corresponding to position 147 along the Kabat EU index or the amino acid corresponding to position 213 along the kappa EU index is replaced with the negatively charged amino acid.

In one preferred embodiment,

i) In the constant domain CL of the first annular fusion polypeptide of the multiple cyclic fusion polypeptide, the amino acid at a position corresponding to position 124 along Kabat is independently selected from the group consisting of lysine (K), arginine (R) or histidine (H) (K) or arginine (R) in the example), and in the constant domain CH1 of the first annular fusion polypeptide of the multicyclic fusion polypeptide, the amino acid at position corresponding to position 147 according to the Kabat EU index, The amino acid at the position corresponding to position 213 according to the index is independently substituted with glutamic acid (E) or aspartic acid (D)

or

ii) In the constant domain CL of the second annular fusion polypeptide of the multiple cyclic fusion polypeptide, the amino acid at a position corresponding to position 124 along Kabat is independently selected from the group consisting of lysine (K), arginine (R) or histidine (H) (K) or arginine (R) in the example), and in the constant domain CH1 of the second annular fusion polypeptide of the multicyclic fusion polypeptide, an amino acid at position corresponding to position 147 along the Kabat EU index, The amino acid at position corresponding to position 213 along the exponent is independently replaced with glutamic acid (E) or aspartic acid (D).

In one embodiment, in the constant domain CL of the second annular fusion polypeptide of the multiple cyclic fusion polypeptide, the amino acid at a position corresponding to positions 124 and 123 along with the kappa EU index is replaced by K.

In one embodiment, in the constant domain CH1 of the second annular fusion polypeptide of the multicyclic fusion polypeptide, the amino acid at positions corresponding to positions 147 and 213 along the Kabat EU index is replaced by E.

In a preferred embodiment, in the constant domain CL of the first annular fusion polypeptide of the multicyclic fusion polypeptide, the amino acid at a position corresponding to positions 124 and 123 according to the kappa EU index is replaced by K, and the first annulus of the multicyclic fusion polypeptide In the constant domain CH1 of the fusion polypeptide, the amino acid at positions corresponding to positions 147 and 213 along the Kabat EU index is replaced by E.

In one embodiment, in the constant domain CL of the second annular fusion polypeptide of the multicyclic fusion polypeptide, the amino acid at a position corresponding to positions 124 and 123 along with the kappa EU index is replaced by K, and the second annular fusion of the multicyclic fusion polypeptide In the constant domain CH1 of the polypeptide, the amino acid at positions corresponding to positions 147 and 213 along the Kabat EU index is substituted with E, and in the variable domain VL of the first annular fusion polypeptide of the multiple cyclic fusion polypeptide, at position 38 along Kabat The amino acid at the corresponding position is substituted with K, and in the variable domain VH of the first annular fusion polypeptide of the multiple cyclic fusion polypeptide, the amino acid at the position corresponding to position 39 along Kabat is replaced with E and the substitution of the multiple cyclic fusion polypeptide In the variable domain VL of the bicyclic fusion polypeptide, the amino acid at the position corresponding to position 38 along Kabat K, and in the variable domain VH of the second annular fusion polypeptide of the multiple cyclic fusion polypeptide, the amino acid at position corresponding to position 39 along Kabat is replaced by E.

In the case of DutaFab, a portion of the binding domain comprises at least the N-terminal fragment (or all) of the antibody heavy chain variable domain (VH) and the first antibody heavy chain constant domain (CHl), and each of the other binding domains comprises an antibody light chain variable (Or all) of the light chain constant domain (VL) and the antibody light chain constant domain (CL), wherein the binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain Wherein the first paratope comprises residues from CDR1 and CDR3 of the VL domain and from CDR2 of the VH domain and the second paratope comprises residues from the CDR1 and CDR3 of the VH domain and from CDR2 of the VL domain ≪ / RTI >

In one embodiment, the first paratope comprises CDR1 and CDR3 of the VL domain and a residue from CDR2 of the VH domain, and the second paratope comprises CDR1 and CDR3 of the VH domain and a residue from CDR2 of the VL domain.

In one embodiment, the heavy chain variable domain of the binding site is based on the human VH3 family heavy chain sequence and the light chain variable domain of the binding site is based on the human Vkappa1 family light chain sequence.

In one embodiment, the heavy chain variable domain of the binding site is based on the human VH3 family heavy chain sequence and the light chain variable domain of the binding site is based on the human Vlambdal family light chain sequence.

III.2. Chimeric and humanized antibody-derived binding sites

In certain embodiments, the binding site of a cyclic fusion polypeptide as reported herein consists of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL). In certain embodiments, the variable domain is a chimeric domain derived from a chimeric antibody, such as a humanized antibody.

&Quot; Framework " or " FR " refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the VH (or VL) sequence as follows: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

Quot; hypervariable region " or " HVR ", as used herein, means that the sequence is hypervariable ("complementarity determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop" Refers to each region of the antibody variable domain comprising an amino acid residue stretch that contains an antigen-contacting residue (" antigen-contacting portion "). Generally, the antibody contains six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

HVR

(a) is present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 A hypervariable loop (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917);

(b) the amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 CDR (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

(c) amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 Antigen contacts (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); And

(d) amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (A), (b), and / or (c), including (H2), 93-102 (H3), and 94-102

.

Unless otherwise indicated, the HVR residues and other residues (e.g. FR residues) of the variable domains are numbered according to Kabat et al. (Homology) herein.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, the non-human antibody is humanized to reduce immunogenicity to humans, but retains the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains from which an HVR, e.g., a CDR (or portion thereof), is derived from a non-human antibody and FR (or a portion thereof) is derived from a human antibody sequence. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues from a non-human antibody (e. G., An antibody from which the HVR residue is derived), for example, antibody specificity or affinity is restored or improved.

Humanized antibodies and methods for their production are described, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and are further described, for example, in the following references: Riechmann, I. et al. , Nature 332 (1988) 323-329; [Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321, and US 7,087,409; [Kashmiri, SV et al., Methods 36 (2005) 25-34] (describing specificity determining region (SDR) grafting); [Padlan, EA, Mol. Immunol. 28 (1991) 489-498) (describing "resurfacing");[Dall'Acqua, WF et al., Methods 36 (2005) 43-60] (describing "FR shuffling"); And Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the " induction screening " approach to FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to, selected framework regions (e. G., Sims, MJ et al. J. Immunol. 151 (1993) 2296-2308); A framework region derived from a common sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (e. G., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 -4289; and Presta, LG et al., J. Immunol. 151 (1993) 2623-2632); (Mature) or human germline framework regions (see, for example, Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633) ; (Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, MJ et al., J Biol. Chem. 271 (19969 22611-22618)).

III.3. A binding site derived from a human antibody

In certain embodiments, the binding site of a cyclic fusion polypeptide as reported herein consists of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL). In certain embodiments, the variable domain is derived from a human antibody.

Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk, M.A. and van de Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce an intact antibody or an intact human antibody having a human variable domain in response to an antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus, either replacing endogenous immunoglobulin loci, or being extrachromosomally or randomly integrated into the chromosome of an animal. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods of obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. For example, to illustrate XENOMOUSE ^TM technology US 6,075,181 and US 6,150,584; US 5,770,429, which describes HuMab® technology; US 7,041, 870, which describes KM MOUSE® technology; US 2007/0061900, which describes VELOCIMOUSE® technology; See WO 2007/131676 which describes an immunological reconstituted mouse. The human variable region derived from an intact antibody produced by such an animal can be further modified.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human xenogene myeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005, ; [Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63]; And [Boerner, P. et al., J. Immunol. 147 (1991) 86-95). Human antibodies produced through human B-cell hybridoma technology are also described in Li, J. et al. , Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Additional methods are described, for example, in US 7,189,826 (which describes the preparation of monoclonal human IgM antibodies from hybridoma cell lines) and [Ni, J., Xiandai Mianyixue 26 (2006) 265-268 Quot ;, which is incorporated herein by reference). Human hybridoma technology (Trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such a variable domain sequence can then be combined with a preferred human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

III.4. Library-derived antibody binding site

In certain embodiments, the binding site of a cyclic fusion polypeptide as reported herein consists of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL). In certain embodiments, a variable domain is isolated by screening a combinatorial library for an antibody having the desired activity or activities.

For example, various methods are known in the art for generating phage display libraries and for screening antibodies against antibodies that possess desirable binding characteristics. These methods have been reviewed, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2001) 1-37, and are described, for example, in McCafferty, J. et al., Nature 348 (1990) 552-554; [Clackson, T. et al., Nature 352 (1991) 624-628]; [Marks, JD et al., J. Mol. Biol. 222 (1992) 581-597; [Marks, JD and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175]; [Sidhu, SS et al., J. Mol. Biol. 338 (2004) 299-310; [Lee, CV et al., J. Mol. Biol. 340 (2004) 1073-1093; [Fellouse, FA, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; And [Lee, CV et al., J. Immunol. Methods 284 (2004) 119-132.

In a constant phage display method, Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455], the repertoires of the VH and VL genes are individually cloned via polymerase chain reaction (PCR) and randomly recombined into a phage library, which is subsequently subjected to antigen-binding phage Can be screened. The phage typically display antibody fragments as short chain Fv (scFv) fragments or Fab fragments. The library from the immunized source provides a high affinity binding site for the immunogen without the need for hybridoma production. Alternatively, Griffiths, A. D. et al. Cloning of natural repertoires (e. g., from human origin) as described in EMBO J. 12 (1993) 725-734 can be used to screen for a wide range of non-magnetic-antigens and also anti- Lt; / RTI > Finally, natural libraries are also described in Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388), cloning of unmodified V-gene segments from stem cells, and the use of a vector containing a random sequence to encode a highly variable CDR3 region and perform rearrangement in vitro Can be synthetically prepared using PCR primers. Patent publications describing human antibody phage libraries are described, for example, in US 5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are referred to herein as human antibodies or human antibody fragments.

IV. Heterogeneous-mass (dimer) somatization domain

Different techniques can be used to confirm correct association of individual cyclic fusion polypeptides to form heterologous-multiresponsive fusion polypeptides. One of them is the so-called "knob-in-hole" operation (see, for example, US Pat. No. 5,731,168). Polycyclic Fusion Polypeptides can also be used to manipulate electrostatic steering effects for the production of Fc-heterodimeric molecules (WO 2009/089004); Cross-linking of two or more annular fusion polypeptides (see, for example, US 4,676,980 and Brennan, M. et al. , Science 229 (1985) 81-83); The use of leucine zippers for the production of double-ring fusion polypeptides (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553).

Several approaches to HC3-modification to support heterodimerization are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

Typically, in the approaches known in the art, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both homologous with the other heavy chain of the same structure, wherein the heavy chain comprising one engineered CH3 domain is no longer homodimerized (For example, the CH3-engineered first heavy chain can no longer be homodimerized with the other CH3-engineered first heavy chain and the CH3-engineered second heavy chain can not be further modified Lt; RTI ID = 0.0 > CH3-engineered < / RTI > second heavy chain). Thus, a heavy chain comprising one engineered CH3 domain is forced to form a heterodimer with another heavy chain comprising a CH3 domain engineered in a complementary manner. In such embodiments, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitutions such that the first and second heavy chains are forced to be heterodimerized, The first and second heavy chains can no longer be homodimerized (e. G., For steric reasons).

Different approaches to support the heavy chain heterodimerization known in the art and incorporated hereinabove and incorporated herein may be combined with a specific amino acid substitution as described above in combination with a "Quot; non-crosslinked Fab region ", and a " cross-linked Fab region " derived from a second antibody that specifically binds to a second antigen, as distinct alternatives used to provide a multispecific antibody as reported herein .

The CH3 domain of a multiple cyclic fusion polypeptide (Contont body) as reported herein is described, for example, in WO 96/027011, [Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; And Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681, which is incorporated herein by reference in its entirety. In this way, the interaction surface of the two CH3 domains is altered to increase the heterodimerization of both the two heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) may be a "knob" while the remainder is a "hole". The introduction of a disulfide bridge further stabilizes the heterodimer (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. 1997) 26-35).

In one preferred embodiment, a multicyclic fusion polypeptide as reported herein comprises a T366W mutation in the CH3 domain of a " knob chain " (i.e., a first annular fusion polypeptide), and a "hole-chain" L368A, Y407V mutants (numbered according to the Kabat EU index) to the CH3 domain of the wild-type polypeptide. Additional interchain disulfide bridges between the CH3 domains can also be used, for example, by introducing a Y349C mutation in the CH3 domain of the " knob chain " and an E356C mutation or S354C mutation in the CH3 domain of the " , et al., Nature Biotech. 16 (1998) 677-681). Thus, in another preferred embodiment, a multicyclic fusion polypeptide as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the E356C, T366S, L368A and Y407V mutations in the other of the two CH3 domains Polycyclic fusion polypeptides, including, or as reported herein, include the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains Additional Y349C mutations in the CH3 domain and additional E356C or S354C mutations in the remaining CH3 domain form a chain interspersed disulfide bridge) (numbered according to Kabat EU index).

However, other knob-in-hole techniques as described in EP 1 870 459A1 may also alternatively or additionally be used. Polycyclic fusion polypeptides as reported herein in one embodiment include the R409D and K370E mutations in the CH3 domain of the " knob chain " and the D399K and E357K mutations in the CH3 domain of the " hole-chain "Quot;).

In one embodiment, a multicyclic fusion polypeptide as reported herein comprises a T366W mutation in the CH3 domain of the " knob chain " and a T366S, L368A and Y407V mutation in the CH3 domain of the " hole chain " The R409D and K370E mutations in the CH3 domain and the D399K and E357K mutations in the CH3 domain of the " hole chain " (numbered according to Kabat EU index).

In one embodiment, a multiple cyclic fusion polypeptide as reported herein comprises a Y349C and T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A and Y407V mutation in the other of the two CH3 domains, Polycyclic fusion polypeptides as reported in the specification have the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains and additionally the CH3 domain of the " knob chain " R409D and K370E mutations and the D399K and E357K mutations in the CH3 domain of the " hole chain " (numbering according to Kabat EU index).

Other techniques are known in the art for modifying the CH3 domain of the heavy chain of a multiple cyclic fusion polypeptide to enhance heterodimerization in addition to " knob-in-the-hole " techniques. These techniques are described in particular in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, 058768, WO 2013/157954 and WO 2013/096291 are contemplated herein as alternatives to the " knob-in-hole technique " in combination with a multiple cyclic fusion polypeptide as reported herein.

In one embodiment of the multicyclic fusion polypeptides as reported herein, the approach described in EP 1870459 is used to support the heterodimerization of the first and second heavy chains of the multicyclic fusion polypeptide. This approach is based on the introduction of a charged amino acid with an opposite charge at the CH3 / CH3-domain-specific amino acid position in the interface between both the first and second heavy chains.

Thus, these embodiments relate to multi-cyclic fusion polypeptides as reported herein wherein the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain in the three-dimensional structure of the antibody are located between the individual antibody CH3 domains Wherein the amino acid sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain each comprise a set of amino acids located within the interface of the three dimensional structure of the cyclic fusion polypeptide, The first amino acid is substituted with the positively charged amino acid and the second amino acid is replaced with the negatively charged amino acid from the amino acid set located at the interface of the CH3 domain of the other heavy chain. A multicyclic fusion polypeptide according to this embodiment is also referred to herein as a " CH3 (+/-) -treated multicyclic fusion polypeptide " (wherein the abbreviation " +/- "Quot;).

Amino acids charged positively in one embodiment of the CH3 (+/-) -treated multicyclic fusion polypeptide as reported herein are selected from K, R and H, and the negatively charged amino acids are E or D .

In one embodiment of the CH3 (+/-) - engineered multicyclic fusion polypeptide as reported herein, the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D Is selected.

In one embodiment of the CH3 (+/-) - engineered multicyclic fusion polypeptide as reported herein, the positively charged amino acid is K and the negatively charged amino acid is E.

In one embodiment of the CH3 (+/-) -treated multicyclic fusion polypeptide as reported herein, the amino acid R at position 409 in the CH3 domain of one heavy chain is substituted with D and the amino acid K at position E And the amino acid D at position 399 in the CH3 domain of the other heavy chain is substituted with K, and the amino acid E at position 357 is substituted with K (numbering according to Kabat EU index).

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2013/157953 is used to support the heterodimerization of the first and second heavy chains of a multiple cyclic fusion polypeptide. In one embodiment of the multicycle fusion polypeptide as reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is D (numbered according to Kabat EU index). In another embodiment of the multicycle fusion polypeptide as reported herein, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced by K, the amino acid L at position 351 is replaced by K, and the other heavy chain CH3 In the domain, the amino acid L at position 351 is substituted with D (numbered according to Kabat EU index).

In another embodiment of the multicycle fusion polypeptide as reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with K, the amino acid L at position 351 is replaced with K, the other heavy chain CH3 In the domain, the amino acid L of position 351 is substituted with D (numbering according to Kabat EU index). In addition, at least one of the following substitutions is included in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is substituted with E, the amino acid Y at position 349 is substituted with D, and the amino acid L at position 368 is substituted with E Numbering according to the EU index). In one embodiment, the amino acid L at position 368 is substituted with E (numbering according to Kabat EU index).

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2012/058768 is used to support heterodimerization of a first annular fusion polypeptide and a second annular fusion polypeptide of a multiple cyclic fusion polypeptide do. In one embodiment of the multicycle fusion polypeptide as reported herein, the amino acid L at position 351 in the CH3 domain of one heavy chain is substituted with Y, the amino acid Y at position 407 is substituted with A, the other CH3 domain of the heavy chain The amino acid T at position 366 is replaced by A, and the amino acid K at position 409 is replaced by F (numbering according to Kabat EU index). In other embodiments, in addition to the above-mentioned substitutions, the positions 411 (native T), 399 (native D), 400 (native S), 405 (native F), 390 At least one of the amino acids of the original K) is substituted (numbering according to Kabat EU index). Preferred substitutions are

Substitution of the amino acid T at position 411 with an amino acid selected from N, R, Q, K, D, E and W (numbering according to Kabat EU index)

Substitution of the amino acid D at position 399 with an amino acid selected from R, W, Y, and K (numbering according to Kabat EU index)

- substitution of the amino acid S of position 400 with an amino acid selected from E, D, R and K (numbering according to Kabat EU index)

- substitution of the amino acid F at position 405 with an amino acid selected from I, M, T, S, V and W (numbering according to Kabat EU index);

- substitution of the amino acid N at position 390 with an amino acid selected from R, K and D (numbering according to Kabat EU index); And

- substitution of the amino acid K at position 392 with amino acids selected from V, M, R, L, F and E (numbered according to Kabat EU index).

In another embodiment of the multiple cyclic fusion polypeptide as reported herein (engineered in accordance with WO 2012/058768), the amino acid L at position 351 in the CH3 domain of one heavy chain is replaced by Y and the amino acid Y at position 407 is A, and the amino acid T at position 366 in the CH3 domain of the other heavy chain is replaced by V, and the amino acid K at position 409 is substituted with F (numbering according to Kabat EU index). In another embodiment of the multicycle fusion polypeptide as reported herein, the amino acid Y at position 407 in the CH3 domain of one heavy chain is substituted with A, and the amino acid T at position 366 in the CH3 domain of the other heavy chain is substituted with A And the amino acid K at position 409 is replaced with F (numbering according to the Kabat EU index). In the last mentioned embodiment, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is substituted with E, the amino acid T at position 411 is substituted with E, the amino acid D at position 399 is substituted with R The amino acid S of position 400 is substituted with R (numbering according to Kabat EU index).

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2011/143545 is used to support heterodimerization of a first annular fusion polypeptide and a second annular fusion polypeptide of a multiple cyclic fusion polypeptide do. In one embodiment of such a polycyclic fused polypeptide as reported herein, amino acid modifications in both the CH3 domain of both heavy chains are introduced at positions 368 and / or 409 (numbering according to Kabat EU index).

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2011/090762 supports the heterodimerization of a first annular fusion polypeptide and a second annular fusion polypeptide of a multiple cyclic fusion polypeptide Is used. WO 2011/090762 relates to amino acid modifications according to the " knob-in-hole " technique. In one embodiment of the CH3 (KiH) -treated multicyclic fusion polypeptide reported herein, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced with W, the amino acid at position 407 in the CH3 domain of the other heavy chain Y is substituted by A (numbering according to Kabat EU index). In another embodiment of the CH3 (KiH) -treated multicyclic fusion polypeptide as reported herein, the amino acid T at position 366 in the CH3 domain of one heavy chain is substituted with Y, and the CH3 domain at position 407 Is substituted with T (numbering according to Kabat EU index).

In one embodiment of the multicyclic fusion polypeptides as reported herein, wherein the IgG2 isotype is disclosed herein, the approach described in WO 2011/090762 supports the heterodimerization of the first and second heavy chains of a multiple cyclic fusion polypeptide .

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2009/089004 supports heterodimerization of a first annular fusion polypeptide and a second annular fusion polypeptide of a multiple cyclic fusion polypeptide Is used. In one embodiment of the multinuclear fusion polypeptide as reported herein, the amino acid K or N at position 392 in the CH3 domain of one heavy chain is a negatively charged amino acid (in one preferred embodiment E or D, In example D), the amino acid D at position 399, the amino acid E or D at position 356, or the amino acid E at position 357 in the CH3 domain of the other heavy chain is substituted with a positively charged amino acid (in one preferred embodiment, K Or R, substituted in one preferred embodiment by K, and in one preferred embodiment, the amino acid at position 399 or 356 is replaced by K) (numbered according to Kabat EU index). In a further embodiment, in addition to the substitutions mentioned above, in the CH3 domain of one heavy chain, the amino acid K or R at position 409 is a negatively charged amino acid (in one preferred embodiment E or D, in one preferred embodiment D ) (Numbering according to Kabat EU index). In a still further embodiment, in addition to or alternatively, the above-mentioned substitutions, in the CH3 domain of one heavy chain, the amino acid K at position 439 and / or the amino acid K at position 370 are independently of one another a negatively charged amino acid E or D in embodiments, D) in one preferred embodiment (numbering according to Kabat EU index).

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2007/147901 supports the heterodimerization of a first annular fusion polypeptide and a second annular fusion polypeptide of a multiple cyclic fusion polypeptide Is used. In one embodiment of the multicycle fusion polypeptide as reported herein, the amino acid K at position 253 in the CH3 domain of one heavy chain is substituted with E, the amino acid D at position 282 is substituted with K, and the amino acid K Is replaced with D, and the amino acid D at position 239 in the other heavy chain CH3 domain is substituted with K, the amino acid E at position 240 is substituted with K, and the amino acid K at position 292 is substituted with D (number according to Kabat EU index Padding).

In one embodiment of a multicyclic fusion polypeptide as reported herein, the approach described in WO 2007/110205 supports the heterodimerization of a first annular fusion polypeptide and a second annular fusion polypeptide of a multiple cyclic fusion polypeptide Is used.

In one embodiment of all aspects and embodiments as reported herein, a multiple cyclic fusion polypeptide is a double cyclic fusion polypeptide or a triple cyclic fusion polypeptide. In one preferred embodiment, the multiple cyclic fusion polypeptide is a bispecific double ring fusion polypeptide.

In one embodiment of all aspects as reported herein, a multicyclic fusion polypeptide has a constant domain structure of an IgG type antibody. In a further embodiment of all aspects as reported herein, a multicyclic fusion polypeptide is characterized in that said multicyclic fusion polypeptide is selected from the Fc-region of human subclass IgGl, or human subclass IgGl with mutations L234A and L235A and optionally P329G ≪ / RTI > region of the Fc region. In one further embodiment of all aspects as reported herein, a multicyclic fusion polypeptide is characterized in that said multicyclic fusion polypeptide comprises the Fc-region of human subclass IgG2. In one further embodiment of all aspects as reported herein, a multicyclic fusion polypeptide is characterized in that said multicyclic fusion polypeptide comprises the Fc-region of human subclass IgG3. In one further embodiment of all aspects as reported herein, a multicyclic fusion polypeptide is characterized in that said multicyclic fusion polypeptide is an Fc-region of human subclass IgG4, or a human having additional mutations S228P and L235E and optionally P329G And the Fc-region of the subclass IgG4.

V. variant

In certain embodiments, amino acid sequence variants of the cyclic fusion polypeptides provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the annular fusion polypeptide. Amino acid sequence variants of the cyclic fusion polypeptide can be prepared by peptide synthesis or by introducing a suitable modification to the nucleotide sequence encoding the cyclic fusion polypeptide. Such modifications include deletion from residues in the amino acid sequence of the annular fusion polypeptide, and / or insertion into and / or substitution thereof. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct retains the desired characteristics, e. G., Antigen-binding.

a) substitution, insertion, and deletion mutants

In certain embodiments, there is provided a cyclic fusion polypeptide variant having one or more amino acid substitutions. Interest regions for inducing replacement mutations include HVR and FR. Conservative substitutions are shown in the table below under the title of " preferred substitutions ". More substantial changes are provided as described further below in the table below under the heading " Exemplary Substitutions " and on the basis of the amino acid side chain class. Amino acid substitutions can be introduced into the annular fusion polypeptide of interest and the product can be screened for the desired activity, for example retention / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be classified according to their common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) Acid: Asp, Glu;

(4) Basicity: His, Lys, Arg;

(5) residues affecting the chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve exchanging members of one of these classes for another.

One type of substitutional variant involves the substitution of one or more hypervariable region residues (e. G., Comprising a binding site derived from humanized or human antibodies) of the parent cyclic fusion polypeptide. In general, the final variant (s) selected for further experiments will have a modification (e. G., Improvement) to certain properties (e. G., Increased affinity, reduced immunogenicity) for the parent cyclic fusion polypeptide /, Or will have substantially retained constant biological properties of the parent cyclic fusion polypeptide. Exemplary substitution mutants can be readily generated using affinity matured annular fusion polypeptides, e. G., Phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant cyclic fusion polypeptides are displayed on phage and screened for specific biological activity (e. G., Binding affinity).

Alterations (e. G., Substitutions) can be performed in the HVR, for example, to improve the affinity of the annular fusion polypeptide. These changes include HVR " hotspots ", or residues encoded by codons that undergo mutations at high frequencies during the somatic cell maturation process (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196) , And / or residues in contact with the antibody, and the final variant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and re-selection therefrom is described, for example, in Hoogenboom, H.R. et al. in Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into a variable gene selected for maturation by any of a variety of methods (e. G., Error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). The secondary library is then created. The library is then screened to identify any cyclic fusion polypeptide variants with the desired affinity. Another way to introduce diversity involves the HVR-designated approach, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

In certain embodiments, substitution, insertion, or deletion can occur within one or more HVRs, so long as such alteration does not substantially reduce the ability of the annular fusion polypeptide to bind to the intended target. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can occur in the HVR. Such alterations may be external to, for example, the antigen-contacting residues in the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains one, two, or three amino acid substitutions.

A useful method for the identification of residues or regions of a cyclic fusion polypeptide that may be a target for mutagenesis is described in Cunningham, B.C. and Wells, J. A., Science 244 (1989) 1081-1085, which is herein incorporated by reference in its entirety. In this method, a group or residue (e.g., charged residues such as arg, asp, his, lys, and glu) of a target residue is identified and a neutral or negatively charged amino acid (e.g., alanine or polyalanine) To determine whether the interaction of its target and the cyclic fusion polypeptide is affected. Additional substitutions can be introduced at the amino acid positions to demonstrate functional sensitivity to initial substitution. Alternatively or additionally, with the crystal structure of the target-cyclic fusion polypeptide complex, the point of contact between the cyclic fusion polypeptide and its target is identified. Such contact residues and neighboring residues may be removed or targeted as candidates for substitution. Variants can be screened to determine whether they contain desirable properties.

Amino acid sequence insertions include sequential insertion of single or multiple amino acid residues, including amino-terminal and / or carboxyl-terminal fusions of a polypeptide length range containing from one residue length to several hundred or more residues. Examples of terminal insertions include cyclic fusion polypeptides having an N-terminal methionyl residue. Other insertional variants of the cyclic fusion polypeptide molecule include fusion of the N-terminal or C-terminal end of the cyclic fusion polypeptide to a polypeptide or enzyme that increases the serum half-life of the cyclic fusion polypeptide (e.g., in the case of ADEPT).

b) glycosylated variant

In certain embodiments, the cyclic fusion polypeptide provided herein is altered to increase or decrease the extent to which the cyclic fusion polypeptide is glycosylated. Addition or deletion of the glycation site can be conveniently performed on the annular fusion polypeptide by altering the amino acid sequence so that more than one glycation site is created or removed.

If the annular fusion polypeptide comprises an Fc-region, the carbohydrate attached thereto may be altered. The native Fc-region containing circular fusion polypeptide typically produced by mammalian cells generally comprises a branched, bi-tactile oligosaccharide attached by an N-linkage to the Asn297 of the CH2 domain of the Fc-region For example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32). Oligosaccharides can include fucose attached to GlcNAc in a " tether " of a bi-tactile oligosaccharide structure, including various carbohydrates such as mannose, N- acetylglucosamine (GlcNAc), galactose, have. In some embodiments, modification of the oligosaccharides in the cyclic fusion polypeptides of the invention can be performed to produce cyclic fusion polypeptide variants with certain improved properties.

In one embodiment, a cyclic fusion polypeptide variant having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc-region is provided. For example, the amount of fucose in such circular fusion polypeptides may be from 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is calculated for a sum of all pools attached to Asn 297 (e.g., complex, hybrid and highly mannose structures) as measured by MALDI-TOF mass spectroscopy, for example as described in WO 2008/077546 , And the average amount of fucose in sugar chains in Asn297. Asn297 refers to an asparagine residue located at approximately position 297 (the EU numbering of Fc-region residues) in the Fc-region, but Asn297 also has about -3% upstream or downstream of position 297 due to a small number of sequence variations in the antibody Amino acids, i. E., Positions 294-300. Such fucosylation variants may have improved ADCC function (see, e.g., US 2003/0157108; US 2004/0093621). Examples of disclosures for " tamifucosyl " or " fucose-deficient " antibody variants are described in US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A. et < RTI ID = 0.0 > al., & J. Mol. Biol. 336 (2004) 1239-1249; [Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines from which furfucosylated cyclic fusion polypeptides can be produced include Lecl3 CHO cells deficient in protein fucosylation (Ripka, J. et al. Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; And WO 2004/056312, especially Example 11), and knockout cell lines such as the alpha-1,6-fucosyltransferase gene,FUT8, Knockout CHO cells (see, for example, Yamane-Ohnuki, N. et al. Biotech. Bioeng. 87 (2004) 614-622; [Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; WO 2003/085107).

The annular fusion polypeptide variant is further provided with a bisected oligo saccharide, for example a bi-tactile oligosaccharide attached to the Fc-region of a cyclic fusion polypeptide is bisected by GlcNAc. Such cyclic fusion polypeptide variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US 6,602,684; And US 2005/0123546. Also provided are cyclic fusion polypeptide variants having at least one galactose residue in an oligosaccharide attached to the Fc-region. Such annular fusion polypeptide variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; And WO 1999/22764.

c) Fc-region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc-region of the annular fusion polypeptide provided herein, thereby generating Fc-region variants. The Fc-region variant may comprise a human Fc-region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention provides a cyclic fusion polypeptide variant (e. G., A fusion protein) that retains some but not all of its effector functions, where in vivo half life is important, but certain effector functions (e. G., Complement and ADCC) are desired candidates for unwanted or deleterious applications . In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the annular fusion polypeptide retains FcRN binding capacity, although the Fc [gamma] R binding is lacking (thereby potentially lacking ADCC activity). The major cell for mediation of ADCC, NK cells, expresses only Fc [gamma] RIIII, whereas monocytes express Fc [gamma] RI, Fc [gamma] RII and Fc [gamma] RIII. FcR expression on hematopoietic cells is described in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. ≪ / RTI > Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in US 5,500,362 (Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059 USA), and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); See US 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays methods may be used is (for example, ACTI ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 ^® Non-Radioactive Cytotoxicity (Promega, Madison, Wis.). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be determined, for example, by the method of Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. ≪ / RTI > The C1q binding assay can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity (see, for example, the C1q and C3c binding ELISAs of WO 2006/029879 and WO 2005/100402) . To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearing / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int. Immunol. 18 (2006: 1759-1769) Reference).

Fc-region containing cyclic fusion polypeptides with reduced effector function include those with substitution of at least one of Fc-region residues 238, 265, 269, 270, 297, 327 and 329 (US 6,737,056). This Fc-region mutant is an Fc-region mutant having substitutions in two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called " DANA " Fc- region mutants with alanine substitutions at residues 265 and 297, Region mutants (US 7,332,581).

Certain Fc-region containing cyclic fusion polypeptide variants with improved or reduced binding to FcR have been described (see, for example, US 6,737,056; WO 2004/056312, and Shields, RL et al. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, the annular fusion polypeptide variant comprises an Fc-region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc-region (EU numbering of residues) .

In some embodiments, for example, in US 6,194,551, WO 99/51642, and in Idusogie, E. et al., J. Immunol. 164 (2000) 4178-4184, alterations are made in the Fc-region that result in altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC).

(Guyer, RL et al., J. Immunol. 117 (1976) 587-593, and Kim, J., et al., Immunotherapy with anti-angiogenic antibodies for improving the binding to the neonatal Fc receptor (FcRn) JK et al., J. Immunol. 24 (1994) 2429-2434) are described in US 2005/0014934. Wherein the antibodies comprise within it an Fc-region having one or more substitutions which improve the binding of the Fc-region to FcRn. These Fc variants have Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 Or 434, e. G., Substitutions of Fc-region residues 434 (US 7,371, 826).

Also, for another example of Fc-region variants, Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; And WO 94/29351.

In one embodiment of all aspects, the cyclic fusion polypeptide comprises (all positions are in accordance with Kabat's EU index):

i) a homodimeric Fc-region of the human IgGl subclass optionally with mutations P329G, L234A and L235A, or

ii) a homodimeric Fc-region of the human IgG4 subclass optionally having the mutations P329G, S228P and L235E, or

iii) a homodimeric Fc-region of the human IgGl subclass optionally having the mutations P329G, L234A, L235A, I253A, H310A, and H435A or optionally with the mutations P329G, L234A, L235A, H310A, H433A and Y436A, or

iv) a heterodimeric Fc-region as described below,

a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

b) one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutants T366S, L368A, Y407V, and S354C, or

c) one Fc-region polypeptide comprises the mutations T366W and S354C, the Fc-region polypeptide comprises a heterodimeric Fc-region comprising the mutations T366S, L368A, Y407V and Y349C,

or

v) all of the Fc-region polypeptides are heterodimeric Fc-regions of the human IgGl subclass comprising the mutations P329G, L234A and L235A,

a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

b) one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutants T366S, L368A, Y407V, and S354C, or

c) one Fc-region polypeptide comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the heterodimeric Fc-region comprising the mutations T366S, L368A, Y407V and Y349C,

or

vi) a heterodimeric Fc-region of a human IgG4 subclass wherein all of the Fc-region polypeptides comprise the mutations P329G, S228P and L235E,

a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

b) one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutants T366S, L368A, Y407V, and S354C, or

c) one Fc-region polypeptide comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the heterodimeric Fc-region comprising the mutations T366S, L368A, Y407V and Y349C,

or

vii) a combination of i), ii), and iii) and one of vi), v) and vi).

In one embodiment of all aspects as reported herein, the cyclic fusion polypeptide comprising the CH3 domain comprises an additional C-terminal glycine-lysine dipeptide (numbered according to G446 and K447, Kabat EU index). In one embodiment of all aspects as reported herein, the cyclic fusion polypeptide comprising the CH3 domain comprises an additional C-terminal glycine residue (numbered according to G446, Kabat EU index).

The cyclic fusion polypeptide as reported herein is characterized in that it is human subclass IgGl, or subclass IgG4, with mutant PVA236, L234A / L235A, and / or GLPSS331 (numbered according to EU index of Kabat) in one embodiment Lt; RTI ID = 0.0 > Fc < / RTI > In a further embodiment, the annular fusion polypeptide comprises an Fc-region of any IgG class, in one embodiment E233, L234, L235, G236, D270, N297, E318, K320, K322, A327, A330, P331 and / Region of the IgG1 or IgG4 subclass, which contains at least one mutation in the amino acid sequence (numbered according to Kabat's EU index). In one further embodiment, the cyclic fusion polypeptide comprises a mutation S228P, or a mutated S228P and L235E (numbered according to Kabat's EU index) (Angal, S., et al., Mol. Immunol. 30 (1993) 105-108) Lt; RTI ID = 0.0 > IgG4 < / RTI > subclass.

The C-terminus of the Fc-region polypeptide contained in the cyclic fusion polypeptide as reported herein can be a complete C-terminal single terminated with the amino acid residue PGK. The C-terminus can be a shortened C-terminal single in which one or both of the C-terminal amino acid residues are removed. In one preferred embodiment the C-terminus is a shortened C-terminus terminated with an amino acid residue PG.

d) Cysteine Manipulated Annular fusion polypeptide

In certain embodiments, it may be desirable to generate a cysteine engineered cyclic fusion polypeptide in which one or more residues are substituted with a cysteine residue. In certain embodiments, the substituted moiety occurs at an accessible site of the cyclic fusion polypeptide. Such that the reactive thiol group is located at an accessible site of the annular fusion polypeptide and the annular fusion polypeptide is conjugated to another moiety such as a drug moiety or linker-drug moiety, as described further herein Can be used to generate the same immunoconjugate. In certain embodiments, a cyclic fusion polypeptide as described herein, particularly any one or more of the following residues of the Containsbodies, can be substituted with cysteine: V205 of a light chain (kappa numbering); A118 of the heavy chain (EU numbering); And S400 (EU numbering) of the heavy chain Fc-region. Cysteine engineered cyclic fusion polypeptides can be produced, for example, analogous to the antibodies described in US 7,521,541.

e) a cyclic fusion polypeptide derivative

In certain embodiments, the cyclic fusion polypeptides provided herein can be further modified to include additional non-monocyclic moieties known in the art and readily available. Suitable moieties for the derivatization of cyclic fusion polypeptides include, without limitation, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, Poly (propylene oxide), poly (propylene oxide), poly (propylene oxide), poly (ethylene oxide), ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly Glycol homopolymers, propyl propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or non-branched. The number of polymers attached to the annular fusion polypeptide can be variable and, if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or types of polymers used in the derivatization are not limited, and include, without limitation, certain characteristics or functions of the cyclic fusion polypeptide to be improved, whether the cyclic fusion polypeptide derivative is to be used in therapy under defined conditions, As shown in FIG.

In another embodiment, conjugates of non-mitotic moieties and cyclic fusion polypeptides that can be selectively heated by radiation exposure are provided. In one embodiment, the non-hydrophobic moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be of any wavelength and includes, without limitation, a wavelength that is not detrimental to normal cells, but which heats the non-hydrophobic moiety to a temperature at which cells close to the antibody-non-hydrophobic moiety are killed.

f) vessel-brain-barrier shuttle junction

In certain embodiments, the cyclic fusion polypeptides provided herein may be further modified to include one or more vascular-brain-barrier shuttle modules known in the art and readily available.

The vascular-brain-barrier shuttle module is characterized by having binding specificity to the vascular-brain-barrier receptor. This binding specificity can be obtained by fusing a vascular-brain-barrier shuttle module to a cyclic fused polypeptide as reported herein, or it can be obtained by introducing a vascular-brain-barrier shuttle module as one of the binding specificities of a multispecific (single or multiple) Can be achieved by introducing binding specificities to the barrier receptor and thus include binding specificity for therapeutic targets and binding specificity for vascular-brain-barrier receptors.

One or more vessel-brain-barrier shuttle modules may be fused to any end of the light or heavy chain of the annular fusion polypeptide as reported herein. In one preferred embodiment, the vascular-brain-barrier shuttle module is fused to the C-terminus of the annular fusion polypeptide. In one preferred embodiment, the vascular-brain-barrier shuttle module is fused to the N-terminus of the annular fusion polypeptide.

One or more vascular-brain-barrier shuttle modules can be fused directly or through a peptide linker to individual annular fusion polypeptides. In one preferred embodiment, the peptide linker has the amino acid sequence GGGGSGGGGS (SEQ ID NO: 64), or GGGGSGGGGSGGGGS (SEQ ID NO: 65) or (G4S) ₆ (SEQ ID NO: 70).

The vascular-brain-barrier shuttle module may be an antibody scFv fragment or Fab. In one embodiment, the vascular-brain-barrier shuttle module is a scFv comprising a light chain variable domain-light chain constant domain-peptide linker-heavy chain variable domain-heavy chain constant domain 1 in N-terminal C-terminal order.

In one embodiment, the vascular-brain barrier shuttle module is a Fab or scFv fragment of a humanized variant of the anti-transferrin receptor-antibody 8D3 with (G4S) ₆ peptide linker (SEQ ID NO: 70).

The term " its humanized variants " refers to the HVR of human and animal 8D3 antibodies on a human framework, optionally randomly introducing one to three mutations into each framework region (FR) and / or hypervariable region (HVR) Means a molecule obtained by grafting.

The heavy chain variable domain has the amino acid sequence of SEQ ID NO: 1, and the anti-transferrin receptor antibody 8D3 (see, for example, Boado, RJ, et al., Biotechnol. Bioeng. 102 (2009) 1251-1258) 71 and the light chain variable domain has the amino acid sequence of SEQ ID NO: 72 (variant 1) or the amino acid sequence of SEQ ID NO: 73 (variant 2).

In one embodiment, the vascular-brain-barrier shuttle module is a Fab or scFv fragment of the humanized variant of the anti-transferrin receptor-antibody 299 (see WO2017 / 055541).

The term " its humanized variants " is intended to mean that the HVR of the 299 antibody is grafted onto a human framework, optionally incorporating one to three mutations in each framework region (FR) and / or hypervariable region (HVR) Means the obtained molecule.

The anti-transferrin receptor antibody 299 is a rabbit antibody wherein the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 74 and the light chain variable domain has the amino acid sequence of SEQ ID NO: 75.

In one embodiment, the vascular-brain-barrier shuttle module comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, 79 or 80; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 82; And (f) a transferrin receptor binding specificity comprising HVR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

In one embodiment, the vascular-brain-barrier shuttle module comprises at least a pair of the heavy chain variable domain of SEQ ID NO: 84 and the light chain variable domain of SEQ ID NO: 85 that form a binding site for the transferrin receptor.

In one embodiment, the vascular-brain-barrier shuttle module comprises at least a pair of a heavy chain variable domain of SEQ ID NO: 86 and a light chain variable domain of SEQ ID NO: 87 that form a binding site for a transferrin receptor.

In one embodiment, the vascular-brain-barrier shuttle module comprises at least a pair of the heavy chain variable domain of SEQ ID NO: 88 and the light chain variable domain of SEQ ID NO: 87 forming a binding site for the transferrin receptor.

In one embodiment, the vascular-brain-barrier shuttle module is a Fab or scFv fragment of a humanized variant of the anti-transferrin receptor-antibody 494 (see EP 15187820).

The term " its humanized variants " is defined by grafting the HVR of the 494 antibody onto a human framework, optionally incorporating one to three mutations in each framework region (FR) and / or hypervariable region (HVR) Means the obtained molecule.

Anti-transferrin receptor antibody 494 is a murine and animal antibody wherein the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 89 and the light chain variable domain has the amino acid sequence of SEQ ID NO: 90.

In one embodiment, the vascular-brain-barrier shuttle module is a Fab or scFv fragment of an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor (huTfR) and the cynomolgus transferrin receptor (cyTfR). In certain embodiments, the anti-transferrin receptor antibody is

- bind to the human transferrin receptor (huTfR) and the cynomolgus transferrin receptor (cyTfR) and /

- has an off-rate to the human transferrin receptor equal to or lower than (i.e., at most) the off-rate of the anti-transferrin receptor antibody 128.1 to the cynomolgus transferrin receptor and the off-rate is determined by surface plasmon resonance And the anti-transferrin receptor antibody 128.1 has the heavy chain variable domain of SEQ ID NO: 91 and the light chain variable domain of SEQ ID NO: 92,

RTI ID = 0.0 > 1 / s < / RTI > to 0.005 < RTI ID = 0.0 > 1 / s.

One aspect as reported herein is < RTI ID = 0.0 >

i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO: 74, and

ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO: 75

An anti-transferrin receptor antibody that specifically binds to a human transferrin receptor and a cynomolgus transferrin receptor,

Wherein the antibody has an off-rate to the human transferrin receptor equal to or lower than (i.e., maximally) off-rate of the anti-transferrin receptor antibody 128.1 to the cynomolgus transferrin receptor,

The off-rate is determined by surface plasmon resonance,

The anti-transferrin receptor antibody 128.1 has the heavy chain variable domain of SEQ ID NO: 91 and the light chain variable domain of SEQ ID NO: 92.

In one embodiment, the antibody has a proline amino acid residue (P) at position 80 in the light chain variable domain (numbering according to Kabat).

In one embodiment, the antibody has an asparagine amino acid residue (N) at position 91 in the light chain variable domain (numbering according to Kabat).

In one embodiment, the antibody has an alanine amino acid residue (A) at position 93 in the light chain variable domain (numbering according to Kabat).

In one embodiment, the antibody has a serine amino acid residue (S) at position 100g in the heavy chain variable domain (numbering according to Kabat).

In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 100g in the heavy chain variable domain (numbering according to Kabat).

In one embodiment, the antibody has an amino acid residue (S) serine at position 65 in the heavy chain variable domain (numbering according to Kabat).

In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (numbering according to Kabat).

In one embodiment, the antibody antibody comprises a proline amino acid residue (P) at position 80 in the light chain variable domain, an asparagine amino acid residue (N) at position 91 in the light chain variable domain, an alanine amino acid residue (A) at position 93 in the light chain variable domain, (S) serine at position 100g in the variable domain, serine amino acid residue (S) at position 65 in the heavy chain variable domain, and glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (numbered according to Kabat) .

In one embodiment, the antibody antibody comprises a proline amino acid residue (P) at position 80 in the light chain variable domain, an asparagine amino acid residue (N) at position 91 in the light chain variable domain, an alanine amino acid residue (A) at position 93 in the light chain variable domain, (Q) at position 100g in the variable domain, a serine amino acid residue (S) at position 65 in the heavy chain variable domain, and a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (numbered according to Kabat) .

One aspect as reported herein is < RTI ID = 0.0 >

i) a heavy chain variable domain (VH) sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 88, and

ii) a light chain variable domain (VL) having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 87;

An anti-transferrin receptor antibody that specifically binds to a human transferrin receptor (huTfR)

Wherein the antibody has about the same off-rate as the antibody comprising the heavy chain variable domain (VH) of SEQ ID NO: 88 and the light chain variable domain (VL) sequence of SEQ ID NO: 87.

In one aspect, the disclosure provides a cyclic fusion polypeptide as reported herein wherein a monovalent binding entity that binds to a vascular-brain-barrier receptor is conjugated via a peptide linker.

In one aspect, the disclosure provides a double-ring fusion polypeptide as reported herein, wherein one cyclic fusion polypeptide is conjugated to a monovalent binding entity that binds to a blood-brain-barrier receptor through a peptide linker.

In one aspect, the present disclosure provides a double-ring fusion polypeptide as reported herein, wherein all of the annular fusion polypeptides are conjugated to a monovalent binding entity that individually binds to a blood-brain-barrier receptor via a peptide linker, do. Such conjugates include two univalent binding entities that bind to the vascular-brain-barrier receptors.

In one embodiment, the monovalent binding entity that binds to the vascular-brain-barrier receptor is selected from the group consisting of proteins, polypeptides, and peptides.

In one embodiment, the monovalent binding entity that binds to the vascular-brain-barrier receptor is selected from the group consisting of vascular-brain-barrier receptor ligands, scFv, Fv, scFab, VHH, in one preferred embodiment scFv or scFab Lt; / RTI >

In one embodiment, the vascular-brain-barrier receptor is selected from the group consisting of a transferrin receptor, an insulin receptor, an insulin-like growth factor receptor, a low density lipoprotein receptor-related protein 8, a low density lipoprotein receptor- ≪ / RTI > In a preferred embodiment, the vascular-brain-barrier receptor is a transferrin receptor.

In one embodiment, the monovalent binding entity that binds to the vascular-brain-barrier receptor is an scFv or one scFab for a transferrin receptor, more particularly a transferrin or a scFv for a transferrin receptor contained within the amino acid sequence of SEQ ID NO: 93, RTI ID = 0.0 > scFv < / RTI > or scFv that recognizes an epitope in the receptor.

In one embodiment, the monovalent binding entity that binds to the vascular-brain-barrier receptor is coupled to the C-terminus of the annular fusion polypeptide by a linker.

In one embodiment, the peptide linker is an amino acid sequence of at least 15 amino acids in length, more preferably 18 to 25 amino acids in length.

In one preferred embodiment, the conjugate comprises a cyclic fusion polypeptide as reported herein (which specifically binds to a brain target) as a brain effector entity, a linker of the sequence GGSGGGGSGGGGSGGGGS (SEQ ID NO: 68), and a vascular brain receptor Wherein the scFab is coupled to the C-terminus of the annular fusion polypeptide by a linker, and the scFab comprises a single scFab as a monovalent binding entity that binds to a human transferrin receptor Recognizes an epitope in the human transferrin receptor contained within the amino acid sequence.

In one preferred embodiment, the conjugate comprises a double-ring fusion polypeptide as reported herein (which specifically binds to a brain target) as a brain effector entity, two linkers of the sequence GGSGGGGSGGGGGSGGGGS (SEQ ID NO: 68) Wherein the scFab is coupled to the C-terminus of a different cyclic fusion polypeptide through one linker, and the scFab is selected from the group consisting of SEQ ID NO < RTI ID = 0.0 > : 93, 94, or 95. < RTI ID = 0.0 >

In one embodiment, the first annular fusion polypeptide comprises a first dimerization module and the second annular fusion polypeptide comprises a second dimerization that enables homodimerization and / or heterodimerization of the two annular fusion polypeptides Module.

In one embodiment, the heterodimerization module of the first annular fusion polypeptide is the knob heavy chain Fc-region and the heterodimerization module of the second annular fusion polypeptide is the hole heavy chain Fc-region (according to the knob-in-the-hole strategy ; Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677 (1996) -681]). The introduction of a disulfide bridge further stabilizes the heterodimer (Merchant, AM, et al., Nat. Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. (1997) 26-35), thereby increasing the yield.

In one embodiment, the homomononisation module of the first annular fusion polypeptide is a heavy chain Fc-region and the homodimerization module of the second annular fusion polypeptide is a heavy chain Fc-region, wherein both Fc-regions comprise amino acid substitutions S364G, L368F, D399K and K409D, wherein the amino acid positions are numbered according to Kabat's EU index. Deformation / mutation maintains the same chain-to-chain attractiveness interaction, but causes repulsion of different chains.

In one embodiment, the heterodimerization module of the first annular fusion polypeptide is a knob heavy chain Fc-region and the heterodimerization module of the second annular fusion polypeptide is a hole heavy chain Fc-region (according to the knob-in- ), Wherein one Fc-region is modified by the amino acid substitutions S364G, L368F, D399K and K409D, where the amino acid positions are numbered according to Kabat's EU index. Deformation / mutation reduces the interchain interactions between the holes and promotes heterodimerization.

&Quot; EU numbering system " or " EU index " is generally used to refer to residues in the immunoglobulin heavy chain constant region (e. G., EU indices are described in Kabat et al., Sequences 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The cyclic fusion polypeptides as reported herein can be used to deliver circular fusion polypeptides across the vascular brain barrier.

In one embodiment, a cyclic fusion polypeptide that is coupled at its C-terminus with a scFab as a monovalent binding entity that binds to a human transferrin receptor has the following structure in the C-terminal direction at the N-terminus:

. Cyclic fusion polypeptides,

. A peptide linker that couples the C-terminus of the annular fusion polypeptide to the N-terminus of the VL domain of the scFab, in one preferred embodiment, the peptide linker has the amino acid sequence GGSGGGGSGGGGSGGGGS (SEQ ID NO: 68)

. The variable light chain domain (VL) and the C-kappa light chain domain of scFab,

. to couple the distal end of C- C- kappa light chain domain of scFab the N- end of the VH domain of scFab peptide linker, in the preferred embodiment the peptide linker is the amino acid sequence _{(G 4 S) 4 GG (} SEQ ID NO: 69 ),

. The variable heavy domain (VH) of the scFab antibody and the IgG CHl light chain domain.

In one embodiment, a cyclic fusion polypeptide that is coupled at its C-terminus with a scFv as a monovalent binding entity that binds to a human transferrin receptor has the following structure in the C-terminal direction at the N-terminus:

. Cyclic fusion polypeptides,

. A peptide linker that couples the C-terminus of the annular fusion polypeptide to the N-terminus of the VL domain of the scFv antibody fragment; in one preferred embodiment, the peptide linker is a peptide having the amino acid sequence GGSGGGGSGGGGSGGGGS (SEQ ID NO: 68)

. The variable light chain domain (VL),

. A peptide linker that couples the C-terminal portion of the variable light chain domain to the N-terminal portion of the VH domain of the scFv, in one preferred embodiment the peptide linker comprises a peptide having the amino acid sequence (G ₄ S) ₄ GG (SEQ ID NO: 69) being,

. The variable heavy domain (VH) of the scFv antibody fragment.

One vessel-brain-barrier shuttle module

In one aspect, the cyclic fusion polypeptide or the multicyclic fusion polypeptide comprises exactly one vascular-brain-barrier binding specificity or shuttle module and is therefore at least bispecific, wherein the vascular-brain-barrier binding specificity or shuttle module

Humanized variants of the variable domains of the anti-human transferrin receptor antibody 8D3 of SEQ ID NO: 71 and 72 or 73, or

A heavy chain variable domain of SEQ ID NO: 88 and a light chain variable domain pair of SEQ ID NO: 87, or

Humanized variants of the variable domains of the anti-human transferrin receptor antibody 494 of SEQ ID NOs: 89 and 90

Lt; / RTI >

Whereby the vascular-brain-barrier binding specificity or shuttle module carries (multi) annular fusion polypeptides across the vascular-brain-barrier.

One or two vessel-brain-barrier shuttle modules

In one aspect, the cyclic fusion polypeptide or the multicyclic fusion polypeptide comprises one or both of the vascular-brain-barrier binding specificities or shuttle module (s) and is therefore at least bispecific, wherein the vascular-brain-barrier shuttle binding site The module is derived from an antibody that binds with low affinity to the vascular-brain barrier receptor (BBB-R, BBB-R binding specificity), and is a vascular-derived from an antibody that binds with low affinity to the vascular- The brain-barrier binding specificity or shuttle module carries (multi) annular fusion polypeptides across the blood-brain-barrier.

In one embodiment, the BBB-R is selected from the group consisting of a transferrin receptor (TfR), an insulin receptor, an insulin-like growth factor receptor (IGF receptor), a low density lipoprotein receptor-related protein 8 (LRP8), a low density lipoprotein receptor- , And heparin-binding epithelial growth factor-like growth factor (HB-EGF). In another such aspect, BBB-R is human BBB-R. In this aspect, BBB-R is TfR. In another such aspect, BBB-R is TfR and the antibody does not inhibit TfR activity. In another such aspect, BBB-R is TfR and the antibody does not inhibit binding of TfR to transferrin.

In one embodiment, the antibody does not compromise binding to one or more of BBB-R and its natural ligand. In one such embodiment, the antibody specifically binds to the human transferrin receptor (hTfR) in a manner that does not inhibit the binding of human transferrin to hTfR.

In one embodiment, the BBB-R binding specificity has an IC ₅₀ for BBB-R from about 1 nM to about 100 μM. In one embodiment, the IC ₅₀ is from about 5 nM to about 100 μM. In one embodiment, the IC ₅₀ is from about 50 nM to about 100 μM. In one embodiment, the IC ₅₀ is from about 100 nM to about 100 μM. In one embodiment, the BBB-R binding specificity has affinity for BBB-R from about 5 nM to about 10 [mu] M. In one embodiment, the BBB-R binding specificity has affinity for BBB-R of about 30 nM to about 1 [mu] M when included in or conjugated to the cyclic fusion polypeptide. In one embodiment, the BBB-R binding specificity has affinity for BBB-R of about 50 nM to about 1 [mu] M when included in or conjugated to the cyclic fusion polypeptide. In one embodiment, the affinity of the annular fusion polypeptide conjugate or BBB-R binding specificity for BBB-R is determined using Scatchard analysis. In one embodiment, the affinity of a cyclic fusion polypeptide conjugate or BBB-R binding specificity to BBB-R is determined using BIACORE analysis. In one embodiment, the affinity of the cyclic fusion polypeptide conjugate or BBB-R binding specificity for BBB-R is determined using competitive ELISA.

Uses of Vascular-Brain-barrier shuttle-containing conjugates

In another embodiment, the disclosure provides a method of increasing exposure of a CNS to a cyclic fusion polypeptide, wherein the cyclic fusion polypeptide is coupled with an antibody or antibody fragment that binds to BBB-R with low affinity, Thereby increasing the exposure of the CNS to the fusion polypeptide.

The term " coupled " includes the case where it is introduced at least as a second binding specificity in a bispecific cyclic fusion polypeptide. The term " coupled " also includes the case where the anti-BBB-R antibody binding specificity is conjugated as an independent binding specificity in the cyclic fusion polypeptide.

In one embodiment, the increase in CNS exposure to the cyclic fusion polypeptide is measured relative to the CNS exposure of the cyclic fusion polypeptide coupled with a typical antibody that does not have a low affinity for BBB-R. In one embodiment, the increase in CNS exposure to the cyclic fusion polypeptide is measured as the ratio of the amount of the cyclic fusion polypeptide identified in the CNS to the amount identified in the post-administration serum. In one embodiment, the increase in CSH exposure produces a rate of greater than 0.1%. In one embodiment, the increase in CNS exposure to the cyclic fusion polypeptide is measured relative to the CNS exposure of the cyclic fusion polypeptide in the absence of the coupled anti-BBB-R antibody. In one embodiment, the increase in CNS exposure to the annular fusion polypeptide is measured through imaging. In one embodiment, an increase in CNS exposure to a cyclic fusion polypeptide is measured by an indirect reading, such as a modification of one or more physiological symptoms.

In a method of increasing retention in the CNS of a cyclic fusion polypeptide administered to a subject, the cyclic fusion polypeptide is conjugated to an antibody or antibody fragment that binds with low affinity to the BBB-R to increase retention in the CNS of the cyclic fusion polypeptide .

In another embodiment, the disclosure provides a method of optimizing the pharmacokinetics and / or pharmacodynamics of a cyclic fusion polypeptide so as to be effective in the CNS of a subject, wherein the cyclic fusion polypeptide comprises an antibody or antibody fragment that binds with low affinity to BBB- So that the affinity of the antibody or antibody fragment for binding to the BBB-R after coupling with the annular fusion polypeptide results in the transport of significant amounts of antibodies or antibody fragments conjugated to the annular fusion polypeptide across the BBB 0.0 > pharmacokinetics < / RTI > and / or pharmacodynamics of the cyclic fusion peptide of the CSN.

In another embodiment, the disclosure provides a method of treating a neurological disorder in a mammal, comprising administering to the BBB-R and treating the mammal with an antibody or antibody fragment coupled to a circular fusion polypeptide, wherein Antibodies are selected to have low affinity for BBB-R to improve CNS uptake of antibodies and coupled cyclic fusion polypeptides. In one embodiment, the treatment results in a reduction or elimination of the disorder symptoms. In another aspect, treatment results in the alleviation of neurological disorders.

In one embodiment of all previous aspects, the anti-BBB-R antibody has an IC ₅₀ for BBB-R from about 1 nM to about 100 μM. In these other embodiments, the IC ₅₀ is from about 5 nM to about 100 μM. In these other embodiments, the IC ₅₀ is from about 50 nM to about 100 μM. In these other embodiments, the IC ₅₀ is from about 100 nM to about 100 μM. In other embodiments, the antibody has affinity for BBB-R from about 5 nM to about 10 [mu] M. In other embodiments, the antibody has affinity for BBB-R from about 30 nM to about 1 [mu] M when coupled with a cyclic fusion polypeptide. In another embodiment, the antibody has affinity for BBB-R from about 50 nM to about 1 [mu] M when coupled with a cyclic fusion polypeptide. In one embodiment, the affinity of an anti-BBB-R antibody or a cyclic fusion polypeptide conjugate for BBB-R is determined using Scatchard analysis. In another embodiment, the affinity of the anti-BBB-R antibody or the cyclic fusion polypeptide conjugate to BBB-R is determined using BIACORE analysis. In another embodiment, the affinity of an anti-BBB-R antibody or a cyclic fusion polypeptide conjugate to BBB-R is determined using competitive ELISA.

In other embodiments, the annular fusion polypeptide conjugate is labeled. In another embodiment, the anti-BBB-R antibody or fragment does not compromise the binding of one or more of BBB-R and its natural ligand. In another embodiment, the anti-BBB-R antibody specifically binds to hTfR in a manner that does not inhibit the binding of human transferrin to hTrF. In another embodiment, the cyclic fusion polypeptide conjugate is administered to the mammal. In another embodiment, the mammal is a human. In other embodiments, the mammal has a neurological disorder. In another embodiment, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, , Peak disease, Paget's disease, cancer, and traumatic brain injury.

Vessel-brain barrier shuttle as a non-shared complex

One part of the non-covalent complex is the vascular brain barrier-shuttle module (BBB-shuttle module), a bispecific antibody with a first binding specificity for hapten and a second binding specificity for vascular-brain barrier receptor (BBBR). This BBB-shuttle module recognizes transcytoseable cell surface targets (e.g., TfR, LRP or other target, BBB-R) on the vascular brain barrier and binds to the occluded hepatocyte fusion polypeptide simultaneously.

More specifically, annular fusion polypeptides are conjugated to haptens and complexed by hapten-binding sites of vascular brain barrier shuttles. Such complexes are clear and stable and specifically deliver looped fusion polypeptides that are hepattened on the vascular brain barrier. Since the haptenized annular fusion polypeptide is complexed non-covalently by a vascular-brain-barrier shuttle, the haptenized annular fusion polypeptide will, on the one hand, have its delivery vehicle (= vessel-brain-barrier shuttle = Specific antibody) and, on the other hand, can be efficiently released after transcytosis. The conjugation with hapten can be carried out without interfering with the activity of the annular fusion polypeptide. Vascular-brain-barrier shuttles do not contain uncommon covalent addition and thus can avoid any immunogenic potential. The complex of a bispecific antibody containing a hapten-specific binding site and a hepattenated cyclic fusion polypeptide imparts a positive biophysical behavior to the cyclic fusion polypeptide. Further, such a complex can target a load on a cell or tissue that displays an antigen recognized by the second binding specificity of the bispecific antibody.

The circular fusion polypeptide is complexed not only by its functionality but also by a vascular-brain-barrier shuttle (= bispecific antibody), despite being haptenized. In addition, the vascular-brain-barrier receptor binding site of the bispecific antibody retains its binding specificity and affinity in the presence of the complexed heptenated cyclic fused polypeptide. Complexes of bispecific antibodies and helptylated cyclic fusion polypeptides as reported herein can be used to target cyclic fusion polypeptides specifically for cells expressing a vascular-brain-barrier receptor. Haptenized annular fusion polypeptides are coupled with bispecific antibodies in a noncovalent manner so that the annular fusion polypeptide can be released after internalization or transcytosis.

VI. Recombinant methods and compositions

Like antibodies can be prepared using recombinant methods and compositions as described, for example, in US 4,816,567. In one embodiment, isolated nucleic acids encoding the circular fusion polypeptides described herein are provided. Such a nucleic acid may encode an amino acid sequence comprising an amino acid sequence comprising one cyclic fused polypeptide and optionally also a second circular fusion polypeptide (e. G., A first circular fusion polypeptide of a double ring fusion polypeptide and a second circular fusion polypeptide Fusion polypeptide). In a further embodiment, one or more vectors (e. G., Expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (1) an amino acid sequence comprising a cyclic fusion polypeptide (homologous or homologous cyclic fusion polypeptide) and optionally a second cyclic fusion polypeptide in the case of a heterodimeric bifunctional fusion polypeptide , And (2) a vector comprising a nucleic acid encoding a second amino acid sequence encoding the amino acid sequence of a further cyclic fusion polypeptide in the case of a multiple cyclic fusion polypeptide, or (2) A first vector comprising a nucleic acid encoding an amino acid sequence comprising a monoclonal fusion polypeptide, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a second annular fusion polypeptide (e. Switched). In one embodiment, the host cell is a eukaryote such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of producing a cyclic fusion polypeptide is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a cyclic fusion polypeptide as provided above under conditions suitable for expression of the cyclic fusion polypeptide , And optionally recovering the cyclic fusion polypeptide from the host cell (or host cell culture medium).

For recombinant production of a circular fusion polypeptide, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell, for example as described above. Such nucleic acids can be readily prepared using conventional procedures.

Suitable host cells for cloning or expression of a circular fusion polypeptide-coding vector include prokaryotic or eukaryotic cells as described herein. For example, cyclic fusion polypeptides can be produced in bacteria, especially when glycation and Fc effector function are not required. See, for example, US 5,648,237, US 5,789,199, and US 5,840,523 for the expression of antibody fragments and polypeptides in bacteria (see also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254). After expression, the annular fusion polypeptide can be isolated from the bacterial cell paste in the aqueous fraction and further purified.

In addition to prokaryotic organisms, eukaryotic microorganisms such as filamentous fungi or yeasts, including fungal and yeast strains, whose glycation pathways are " humanized " and consequently produce cyclic fusion polypeptides with partial or full human glycation patterns (Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; Li, H. et al., Nat. Biotech. 24 (2006) 210-215 ).

Suitable host cells for the expression of glycated cyclic fusion polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, for the transfection of Spodoptera frugiperda cells, a number of baculovirus strains which can be used in combination with insect cells have been identified.

Plant cell cultures can also be used as hosts (see, for example, US Pat. No. 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429, which describe PLANTIBODIES ^TM technology for antibody production in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 (COS-7) transformed with SV40; Human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); Fetal hamster kidney cells (BHK); Mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); Monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); Human cervical carcinoma cells (HELA); Canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); Human lung cells (W138); Human liver cells (Hep G2); Mouse mammary tumor (MMT 060562); See, e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; And FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), including DHFR ^- CHO cells; And myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of a suitable mammalian host cell line suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268].

VII. Assay

The circular fusion polypeptides provided herein can be used to identify, screen for, or identify their physical / chemical properties and / or biological activity by a variety of assays known in the art to determine the binding of the polypeptide and its target Feature can be identified.

VIII. Immunoconjugate

The present invention also relates to a pharmaceutical composition comprising one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) An immunoconjugate comprising a cyclic fusion polypeptide as reported herein conjugated to an isotope.

In one embodiment, the immunoconjugate comprises a cyclic fused polypeptide, without limitation, a mytansinoid (see US 5,208,020, US 5,416,064 and EP 0 425 235 B1); Auristatin such as monomethylauristatin drug moiety DE and DF (MMAE and MMAF) (see US 5,635,483, US 5,780,588, and US 7,498,298); Dolastatin; 53 (1993) 3336-3342 (1993), which is incorporated herein by reference in its entirety. And Lode, HN et al., Cancer Res. 58 (1998) 2925-2928); Anthracycline such as daunomycin or doxorubicin (Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523); [Jeffrey, SC et al., Bioorg. Med. Chem. Lett. (2006) 358-362]; [Torgov, MY et al., Bioconjug. Chem. 16 (2005) 717-721]; [Nagy, A. et al., Proc Natl Acad Sci USA 97 [King, HD et al., J. Med. Chem. 45 (2002) 4336-834]; [Dubowchik, GM et al., Bioorg. & Med. Chem. Letters 12 Conjugated polypeptide conjugated to one or more drugs including methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, laurotaxel, tefluthell, and alltaxel; tricothecen; and CC1065; Drug conjugate.

In other embodiments, the immunoconjugate is selected from the group consisting of a diphtheria A chain, an unbound active fragment of a diphtheria toxin, an exotoxin A chain (from Pseudomonas aeruginosa), a lysine A chain, an Abrin A chain, , Alpha-sarcine, Aleurites fordii protein, dianthine protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), moordica charantia inhibitor, An enzymatically active toxin or fragment thereof, including an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an antioxidant, a catechin, a catechin, a sapaonaria officinalis inhibitor, gellonin, mitogelin, Lt; RTI ID = 0.0 > fusion < / RTI > polypeptide as reported herein.

In another embodiment, the immunoconjugate comprises a cyclic fusion polypeptide as reported herein that is conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioactive isotopes are available for the generation of radioactive conjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu. When a radioactive conjugate is used for detection, it is used as a spin label for Sintigraphic experimental radioactive atoms, e.g. TC ^99m or I ¹²³ , or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI) Iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of cyclic fusion polypeptides and cytotoxic agents include various dihydric protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl- Imidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (P-diazonium benzoyl) -ethylenediamine), diisocyanates (e.g., bis-diazonium salts), bis- Toluene 2,6-diisocyanate), and bis-activated fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins are described in Vitetta, E.S. et al., Science 238 (1987) 1098-1104. Carbon-14-labeled 1-isothiocyanato-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to a cyclic fusion polypeptide (WO 94/11026 Reference). The linker may be a " cleavable linker " that facilitates release of the cytotoxic drug from the cell. (Chari, RV et al., Cancer Res. 52 (1992) 127-131; U.S. Pat. No. 5,208,020) may be used as the linker, for example, as an acid-labile linker, a peptidase-sensitive linker, a light labile linker, a dimethyl linker or a disulfide- Can be used.

Immunoconjugates herein are commercially available, including without limitation SMBS, SMBB, SMPH, SBP, SIB, SBAB, SIA, SIAB, SMCC, GMBS, HBVS, LC-SMCC, MBS, MPBH, (S), including sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIB, sulfo- SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfonyl) benzoate) Lt; RTI ID = 0.0 > of-linker agent. ≪ / RTI >

IX. Methods and compositions for diagnosis and detection

In certain embodiments, any of the annular fusion polypeptides provided herein are useful for detecting the presence of a target of interest in a biological sample. As used herein, the term " detecting " encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises blood, serum, plasma, cells or tissue.

In one embodiment, a cyclic fusion polypeptide is provided for use in a diagnostic or detection method. In a further aspect, a method is provided for detecting the presence of a target of a cyclic fusion polypeptide in a biological sample. In certain embodiments, the method comprises the steps of contacting a biological sample with a cyclic fusion polypeptide as described herein under conditions that allow binding of the target with a cyclic fusion polypeptide, and contacting the biological sample with a cyclic fusion polypeptide and a target thereof, Is formed. Such methods may be in vitro or in vivo methods. In one embodiment, a cyclic fusion polypeptide is used to select an eligible subject for therapy with the cyclic fusion polypeptide.

In certain embodiments, labeled cyclic fusion polypeptides are provided. A label may be a directly or indirectly detected moire (e. G., Via fluorescent or luminescent, colorimetric, electron-dense, chemiluminescent, and radioactive labels) Such as enzymes or ligands. Exemplary labels include, but are not limited to, radioactive isotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophore such as rare earth chelate or fluorescein and derivatives thereof, rhodamine and derivatives thereof, , Luciferase, such as firefly luciferase and bacterium luciferase (US 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, beta -galactoside Glucoamylase, lysozyme, saccharide oxidase such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dihydrogenase, heterocycle oxidase such as wokase and xanthine oxidase (dye precursors such as HRP , Lactoperoxidase, or an enzyme that applies hydrogen peroxide to oxidize microperoxidase), biotin / avidin, spin label, bacteriophage label, stable It includes organic radicals and the like.

X. Pharmaceutical Formulation

Pharmaceutical formulations of a cyclic fusion polypeptide as described herein are prepared by mixing such a cyclic fusion polypeptide having a desired degree of purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or aqueous solution Remington ' s Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, without limitation, buffering agents such as phosphate, citrate, and other organic acids; Antioxidants including ascorbic acid and methionine; A preservative such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol ; 3-pentanol; and m-cresol); Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as poly (vinylpyrrolidone); Amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt formation counterion such as sodium; Metal complexes (e. G., Zn-protein complexes); And / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rhuPH20 ( HYLENEX ( ^R) , Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rhuPH20, are described in US 2005/0260186 and US 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, the latter formulation comprising a histidine-acetate buffer.

Formulations herein may also contain more than one active ingredient, if desired for the particular indication being treated, preferably those having complementary activity that do not adversely affect one another. These active ingredients are suitably present in combination in amounts effective for their intended purpose.

The active ingredient may be, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, Can be entrapped in drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. This technique is disclosed in Remington ' s Pharmaceutical Sciences, 16 ^th edition, Osol, A. (ed.) (1980).

Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing cyclic fusion polypeptides, which are in the form of molded articles, for example in the form of films or microcapsules.

Formulations used for in vivo administration are generally sterile. Sterilization can be easily accomplished, for example, by filtration through a sterile filter membrane.

XI. Therapeutic methods and compositions

Any of the annular fusion polypeptides provided herein can be used in therapeutic methods.

In one aspect, a cyclic fusion polypeptide is provided for use as a medicament. In a further aspect, there is provided a cyclic fusion polypeptide for use in treating a disease. In certain embodiments, there is provided a cyclic fusion polypeptide for use in a therapeutic method. In certain embodiments, the invention provides a cyclic fusion polypeptide for use in a method of treating a subject having a disease comprising administering to the subject an effective amount of a cyclic fusion polypeptide. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. According to any of the above embodiments, the " entity " is preferably a human.

In a further aspect, the invention provides the use of a cyclic fusion polypeptide in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for the treatment of a disease. In a further embodiment, the medicament is for use in a method of treating a disease comprising administering an effective amount of a medicament to a subject having the disease. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. According to any of the above embodiments, the " entity " may be a human.

In a further aspect, the invention provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease an effective amount of a cyclic fusion polypeptide. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. According to any of the above embodiments, the " entity " may be a human.

In a further aspect, the invention provides a pharmaceutical formulation comprising any of the cyclic fusion polypeptides provided herein, for use, for example, in any of the above therapeutic methods. In one embodiment, the pharmaceutical formulations comprise any of the cyclic fusion polypeptides provided herein and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical formulations comprise any of the cyclic fusion polypeptides provided herein and at least one additional therapeutic agent.

The cyclic fusion polypeptides as reported herein can be used alone or in combination with other agents of therapy. For example, a cyclic fusion polypeptide of the invention may be co-administered with at least one additional therapeutic agent.

Such combined therapies referred to above encompass combination administration (where two or more therapeutic agents are included in the same or separate formulations), and individual administration, and in such cases administration of the cyclic fusion polypeptide of the invention may be accomplished by administration of additional therapeutic agents or agents Before, concurrently with, and / or after administration of the compound. In one embodiment, administration of the cyclic fusion polypeptide and administration of the additional therapeutic agent are within about one month of each other, or within about 1 week, 2 weeks or 3 weeks, or about 1 day, 2 days, 3 days, 4 days, 5 days Or within six days. Suitable cyclic fused polypeptides of the invention may also be used in combination with radiation therapy.

The annular fusion polypeptides (and any additional therapeutic agents) of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired, localized administration for lesions. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosage may be by intravenous or subcutaneous injection, depending on any suitable route, for example injection, e.g. whether partial administration is short-lived or chronic. A variety of dosing schedules are contemplated herein, including, without limitation, single or multiple dosing at various times, bolus dosing, and pulse injection.

The cyclic fusion polypeptides of the present invention are formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the schedule of administration, and other factors known to the practitioner do. The cyclic fusion polypeptide need not be, but is optionally formulated with one or more agents currently used to prevent or treat a subject disorder. The effective amount of such other agent depends on the amount of the cyclic fusion polypeptide present in the formulation, the type of disorder or treatment, and other factors described above. They are generally used in the same dosage and route of administration as described herein, or by any route determined to be experimentally / clinically appropriate, at about 1 to 99%, or at any dosage, of the doses described herein Is used.

(For use alone or in combination with one or more other therapeutic agents) of the cyclic fusion polypeptides of the present invention for the prevention or treatment of disease will depend on the type of disease to be treated, the type of cyclic fusion polypeptide, the severity and course of the disease, Whether the cyclic fusion polypeptide is administered for prophylactic or therapeutic purposes, the previous therapy, the clinical history of the patient and the response to the cyclic fusion polypeptide, and the judgment of the observer. The cyclic fusion polypeptide is suitably administered to the patient during one or a series of treatments. Depending on the type and severity of the disease, a cyclic fusion polypeptide of about 1 μg / kg to 15 mg / kg (eg, 0.5 mg / kg to 10 mg / kg) may be administered, for example, This may be the initial candidate dose for administration to the patient. One typical daily dose may range from about 1 [mu] g / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration for several days or more, depending on the condition, the treatment is generally continued until a favorable inhibition of the disease symptoms occurs. One exemplary dose of the cyclic fusion polypeptide can range from about 0.05 mg / kg to about 10 mg / kg. Thus, a dose of at least about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to a patient. Such a dose may be administered intermittently, e. G. Every week or every three weeks (e. G., The patient receives from about 2 to about 20, or for example about 6 doses of cyclic fusion polypeptide). Initially, one or more lower doses may be administered after a higher loading dose. However, other capacity planning can be useful. The progress of these therapies is easily monitored through conventional techniques and assays.

It is understood that any of the above formulations or methods of treatment may be performed using the immunoconjugates of the invention in addition to or in place of the cyclic fusion polypeptide.

XII. Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and / or diagnosis of disorders. The article of manufacture comprises a package insert or label on or associated with the container and container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags and the like. The container may be formed of various materials such as glass or plastic. The container may be combined with other compositions that are effective in treating, preventing, and / or diagnosing the condition, or may be provided with a sterile access port that holds the composition alone (e.g., the container may be an intravenous solution, It may be a vial with a stopper that can be pierced with a needle). At least one active agent in the composition is a cyclic fused polypeptide of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. In addition, the article of manufacture comprises: (a) a first container comprising a composition therein, wherein the composition comprises a cyclic fused polypeptide of the invention; And (b) a second container containing the composition therein, wherein the composition further comprises a cytotoxic agent or other therapeutic agent. The article of manufacture in such embodiments of the present invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as a spent bacterial (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution . But may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

All references (science, books, or patents) cited herein are incorporated by reference.

The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be understood that modifications may be made to the procedures described without departing from the spirit of the invention.

Brief Description of Drawings

Figure 1: Schematic representation of the general structure of a cyclic fusion polypeptide reported herein.

Figure 2: Schematic representation of the general structure of the double-ring fusion polypeptides reported herein.

Figure 3: Schematic representation of the general structure of the triple annular fusion polypeptide reported herein.

Figure 4: Schematic representation of the general structure of a quadruplex fusion polypeptide reported herein.

Figure 5: Angular alignment and spatial distance between the binding sites of exemplary double ring fusion polypeptides and common IgG type antibodies reported herein.

Figure 6: Analysis of product quality of cyclic fusion polypeptides reported herein by mass spectrometry.

Figure 7: Differential effects of each trastuzumab and double and quadruple anti-Her2 contoll bodies associated with proliferation.

Figure 8: Binding to an FcRn of an exemplary double ring fusion polypeptide reported herein.

Figure 9: ADCC kinetics of the anti-Her2 con- tents body and trastuzumab.

10 + 11: Schematic representation of the orientation and relative position of the VH domain of the "VH-in" orientation (close to the Fc-region) and the "VH-out" orientation (further away from the Fc-region).

12: An exemplary chain of individualized anti-cMET cyclic fusion polypeptides used in the manufacture of the bispecific double ring fusion polypeptides reported herein.

Figure 13: A double ring fusion polypeptide reported herein wherein one annular fusion polypeptide comprises a VH / VL- pair as a binding site and the remaining annular fusion polypeptide comprises a peptide-loaded MHC-I complex as a binding site.

Figure 14: Effect based on MHC-I mediated cell mobilization and cell clearance of the double-ring fusion polypeptides and MHC-I IgG-type antibody fusion reported in this specification and shown in Figure 13.

Figure 15: UV absorption chromatogram (280 nm) in the form of different con- tents bodies.

XIII. Example

The following are examples of the methods and compositions of the present invention. In view of the general description provided above, it is understood that various other embodiments may be practiced.

Materials and methods

Recombinant DNA technology

Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Molecular biology reagents were used according to the manufacturer's instructions.

Genes and Oligonucleotide Synthesis

Preferred gene fragments were prepared by chemical synthesis in Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were amplified for propagation / amplification. Lt; / RTI > plasmid. The DNA sequence of the subcloned gene fragment was verified by DNA sequencing. Alternatively, short synthetic DNA fragments may be assembled by PCR or by annealing the chemically synthesized oligonucleotides. Individual oligonucleotides were prepared from metabion GmbH (Planegg-Martinsried, Germany).

reagent

All commercially available chemicals, antibodies and kits were used as provided in accordance with the manufacturer's protocol unless otherwise specified.

Example  One

Construction of expression plasmids for cyclic fusion polypeptides

For expression of a circular fusion polypeptide as reported herein, a transcription unit comprising the following functional elements was used:

- immediate early enhancers and promoters from human cytomegalovirus (P-CMV) containing intron A,

Human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

- animal and immunoglobulin heavy chain signal sequence,

- a nucleic acid encoding an individual annular fusion polypeptide, and

- bovine growth hormone polyadenylation sequence (BGH pA).

In addition to the expression unit / cassette comprising the desired gene to be expressed, the basic / standard mammalian expression plasmid contains

- This. Origin of replication originating from the vector pUC18 which enables replication of this plasmid in E. coli, and

- This. Beta-lactamase gene that confers resistance to cola on ampicillin

Lt; / RTI >

Example  2

Expression of a cyclic fusion polypeptide

Transient expression of the cyclic fusion polypeptide was performed in suspension-adapted HEK293F (FreeStyle 293-F cells; Invitrogen) cells using the transfection reagent 293-free (Novagen).

Cells were thawed in a 125 mL shaking flask, then diluted (at least 4 times) and passed (30 mL volume) (37 C, 7% CO ₂ , 85% humidity, incubation / shaking at 135 rpm).

The cells were expanded to 250 mL volume with 3x10 ⁵ cells / mL. After 3 days, the cells were split and freshly seeded at a density of 7 * 10 < ⁵ > cells / mL in a 250 mL volume in a 1 liter shake flask. Will be transfected after 24 hours at a cell density of approximately 1.4-2.0x10 ⁶ cells / mL.

Prior to transfection, 250 [mu] g plasmid-DNA was diluted to a final volume of 10 mL using pre-heat (water bath; 37 [deg.] C) Opti-MEM (Gibco). The solution was carefully mixed and incubated at room temperature for no longer than 5 minutes. Then 333.3 [mu] l of 293-preformed transfection reagent was added to the DNA-OptiMEM-solution. The solution was then carefully mixed and incubated at room temperature for 15-20 minutes. The total volume of the mixture was added to a 250 mL HEK-cell-culture-volume 1 L shake flask.

Incubation / shaking was performed at 37 ° C, 7% CO ₂ , 85% humidity, 135 rpm for 6 days or 7 days.

The supernatant was harvested through a first centrifugation-step at 2,000 rpm, 4 ° C, for 10 minutes. The supernatant was then transferred to a new centrifuge-flask for secondary centrifugation at 4,000 rpm, 4 ° C for 30 minutes. Subsequently, the cell-free-supernatant was filtered through a 0.22 μm bottle-top-filter and stored in the freezer (-20 ° C.).

Example  3

Purification of cyclic fusion polypeptides

The antibody-containing culture supernatant was filtered and purified through two chromatographic steps. Use of the antibody _{PBS (1 mM KH 2 PO 4} , 10 mM Na 2 HPO 4, 137 mM NaCl, 2.7 mM KCl), HiTrap MabSelectSuRe (GE Healthcare) were equilibrated with pH 7.4 was trapped through chemical conversion chromatography friendly. Unbound protein was removed by washing with equilibration buffer, and the antibody was recovered in 50 mM citrate buffer, pH 2.8, immediately after which the eluate was neutralized to pH 6.0 using 1 M Tris-base, pH 9.0. Size exclusion chromatography on Superdex 200 ^TM (GE Healthcare) was used as the second purification step. Size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody containing solution was concentrated using an Ultrafree-CL centrifuge filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass.) And stored at -80 ° C.

Example  4

Binding of anti-Her2 cyclic fusion polypeptide

Surface plasmon resonance Her2 receptor binding

Chip Surface: CM5-Chip

T: 37 ° C and 25 ° C for assay settings 1 and 2, respectively

Running buffer : PBS + 0.05% (v / v) Tween 20

Dilution buffer : running buffer + 0.1% BSA

Analyte: c (HER2 ECD) = control for assay set 1 0.41-900 nM;

c (dimer anti-Her2 control body) = 3.7-300 nM,

c (tetramer anti-Her2 constant body) = 1.85-150 nM,

c (trastuzumab) = 3.7-300 nM for assay setting 2

Ligand: dimer and tetramer bound through an anti-human Fc-region antibody for assay setting 1, Her2 con- ttus body, trastuzumab; In the case of Assay Setting 2, Her2-ECD bound via putuzumab.

The reaction unit is directly proportional to the molecular weight. The theoretical maximum value of the analyte binding is based on the known binding level of the Her2 ECD. 100% = 1 molecular dimer The anti-Her2 contusbodies or trastuzumab are each bound to two molecules HER2 ECD; One molecular tetramer anti-Her2 con- ttus body binds to the four-molecule HER2 ECD.

Example  5

ADCC Assay - ACEA

BT-474 cells were " solubilized " with Accutase and counted in medium to a cell density of 2x10E5 cells / mL. 50 [mu] l of the medium was pipetted in each well of a 96-well plate and the background effect was measured in ACEA and finally a 50 [mu] l cell suspension / well (= 10,000 cells / well) was added. Plates were placed in ACEA and cell counts were determined after 15 minutes. Subsequently, the medium was removed by pipetting, the washing step was performed with AIM-V medium, and three different concentrations of 50 [mu] l antibody were added. NK cells were counted and placed in AIM-V at a cell density of 6x10E5. 50 占 퐇 (30,000 cells / well) ad E / T3: 1 was added to ACEA and the cell index was measured every 5 minutes. After 24 hours, the experiment was stopped and the ADCC was calculated at 2 and 4 hours.

Example  6

Mass spectrometric analysis of dimeric anti-Her2 cyclic fusion polypeptides

PNGase F was obtained from Roche Diagnostics GmbH (14.3 U / L; solution in sodium phosphate, EDTA and glycerol). Proteases that specifically cleave in the hinge region of the IgG antibody were freshly reconstituted from the lyophilisate prior to digestion.

Enzymatic desalting by PNGase F

50 [mu] g of Contoll's body was dissolved in 10 mM sodium phosphate buffer, pH 7.1 to a final concentration of 0.6 mg / mL Diluted, and desferred for 16 hours at 37 DEG C using 1 [mu] l of PNGase F. [

Enzymatic cleavage

The desaturated sample was diluted with 200 mM Tris buffer, pH 8.0 to a final concentration of 0.5 mg / mL Diluted, and then digested with IgG specific protease at 37 DEG C for 1 hour.

ESI-QTOF mass spectrometry

Samples were desalted by HPLC on a Sephadex G25 column (Kronlab, 5x250mm, TAC05 / 250G0-SR) using 40% acetonitrile and 2% formic acid (v / v). The total mass was determined via ESI-QTOF MS (Bruker Daltonik) on the maXis 4G UHR-QTOF MS system equipped with a TriVersa NanoMate source (Advion). Calibration was performed using sodium iodide (Waters ToF G2-Sample Kit 2 Part: 700008892-1). For digested Contes bodies, data were acquired at 1000-4000 m / z (ISCID: 30 eV). The raw mass spectra were evaluated and converted to individual relative molar masses. For visualization of the results, a deconvoluted mass spectrum was generated using proprietary software.

                         SEQUENCE LISTING <110> F. Hoffmann-La Roche AG   <120> The Contorsbody - a single chain target binder <130> P33504-WO <150> EP16167920 <151> 2016-05-02 <160> 105 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Arg-tag <400> 1 Arg Arg Arg Arg Arg 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Arg-tag 2 <400> 2 Arg Arg Arg Arg Arg 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> His-tag <400> 3 His His His His His 1 5 <210> 4 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 4 Lys Asp His Leu Ile His Asn Val His Lys Glu Phe His Ala His Ala 1 5 10 15 His Asn Lys              <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 5 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 6 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 6 Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr 1 5 10 15 Lys Asp Asp Asp Asp Lys             20 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag # <400> 7 Ala Trp Arg His Pro Gln Phe Gly Gly 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 8 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 9 Met Asp Val Glu Ala Trp Leu Gly Ala Arg 1 5 10 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 10 Met Asp Val Glu Ala Trp Leu Gly Ala Arg Val Pro Leu Val Glu Thr 1 5 10 15 <210> 11 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 11 Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 1 5 10 15 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro             20 25 30 Gln Gly Gln Arg Glu Pro         35 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 12 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 13 Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser 1 5 10 15 <210> 14 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 14 Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg 1 5 10 15 Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu             20 25 <210> 15 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> amino acid tag <400> 15 Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser His Tyr 1 5 10 15 Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser             20 25 30 Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu         35 40 45 <210> 16 <211> 32 <212> PRT <213> Butyrivibrio fibrisolvens <400> 16 Met Asp Trp Asn Ala Asn Ile Ala Pro Gly Asn Ser Val Glu Phe Gly 1 5 10 15 Ile Gln Gly Aly Gly Ser Val Gly Asn Val Ile Asp Ile Thr Val Glu             20 25 30 <210> 17 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> chitin-binding-domain <400> 17 Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala 1 5 10 15 Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln Pro             20 25 30 His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu Trp         35 40 45 Gln Leu Gln     50 <210> 18 <211> 209 <212> PRT <213> Chondrus crispus <400> 18 Met Pro Glu Ile Lys Leu Thr Tyr Phe Asp Met Arg Gly Arg Ala Glu 1 5 10 15 Ala Ser Arg Leu Ala Leu Val Val Gly Glu Ile Pro Phe Glu Asp Glu             20 25 30 Arg Val Val Phe Asp His Trp Lys Glu Ala Lys Pro Lys Thr Pro Tyr         35 40 45 Ala Ala Leu Pro Met Leu Thr Val Asp Gly Met Gln Val Ala Gln Ser     50 55 60 Asp Ala Ile Leu Arg Tyr Cys Gly Lys Leu Ala Gly Leu Tyr Pro Ser 65 70 75 80 Asp Pro Leu Glu Ala Ala Lys Val Asp Glu Val Gly Gly Val Ile Asp                 85 90 95 Asp Val Thr His Ala Met Tyr Arg Tyr Arg Gly Asp Asp Lys Asp Lys             100 105 110 Leu Arg Glu Glu Arg Asp Lys Phe Ser Lys Val Asp Val Pro Arg Tyr         115 120 125 Val Gly Ala Leu Glu Lys Arg Leu Glu Ala Phe Gly Asp Gly Pro Trp     130 135 140 Ala Val Gly Gly Asn Met Thr Ile Ala Asp Leu His Ile Cys His Leu 145 150 155 160 Val Thr Asn Ile Arg Cys Gly Met Leu Asp Phe Val Asp Lys Asp Leu                 165 170 175 Leu Glu Gly Tyr Val Arg Ile Val Lys Ser Tyr Ser Ala Val Met Glu             180 185 190 His Pro Lys Val Thr Glu Trp Tyr Glu Lys Lys Pro Val Lys Met Phe         195 200 205 Ser      <210> 19 <211> 396 <212> PRT <213> Escherichia coli <400> 19 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys             20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu         35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu     50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr                 85 90 95 Ala Gln Ser Gly Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln             100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys         115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn     130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn                 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly             180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly         195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu     210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp                 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val             260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu         275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu     290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu                 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys             340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala         355 360 365 Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp     370 375 380 Glu Ala Leu Lys Asp Ala Gln Thr Arg Ile Thr Lys 385 390 395 <210> 20 <211> 98 <212> PRT <213> Homo sapiens <400> 20 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val          <210> 21 <211> 107 <212> PRT <213> Homo sapiens <400> 21 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             20 25 30 Trp Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     50 55 60 Glu Ser Thr Tyr Arg Trp Ser Val Leu Thr Val Leu His Gln Asp Trp 65 70 75 80 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro                 85 90 95 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             100 105 <210> 22 <211> 106 <212> PRT <213> Homo sapiens <400> 22 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe             20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu         35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe     50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr                 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly             100 105 <210> 23 <211> 10 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE &Lt; 222 > (8) &Lt; 223 > X = S or P <400> 23 Asp Lys Thr His Thr Cys Pro Xaa Cys Pro 1 5 10 <210> 24 <211> 7 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE &Lt; 222 > (5) &Lt; 223 > X = S or P <400> 24 His Thr Cys Pro Xaa Cys Pro 1 5 <210> 25 <211> 5 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE &Lt; 222 > (3) &Lt; 223 > X = S or P <400> 25 Cys Pro Xaa Cys Pro 1 5 <210> 26 <211> 330 <212> PRT <213> Homo sapiens <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 27 <211> 330 <212> PRT <213> Homo sapiens <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 28 <211> 326 <212> PRT <213> Homo sapiens <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro             100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn                 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp             180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro         195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu     210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys                 325 <210> 29 <211> 377 <212> PRT <213> Homo sapiens <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro             100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg         115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys     130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr         195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln             260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met         275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu                 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile             340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln         355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys     370 375 <210> 30 <211> 327 <212> PRT <213> Homo sapiens <400> 30 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys                 325 <210> 31 <211> 226 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE &Lt; 222 > (131) .. (131) <223> X = E or D <220> <221> MISC_FEATURE <133> (133) <223> X = M or L <220> <221> misc_feature 136, 136, <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature (138). (138) <223> Xaa can be any naturally occurring amino acid <400> 31 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 32 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a hole mutation <400> 32 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 33 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a knob mutation <400> 33 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 34 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with the mutations        L234A, L235A <400> 34 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 35 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a L234A, L235A        and hole mutation <400> 35 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 36 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a L234A, L235A        and knob mutation <400> 36 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 37 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a L234A, L235A        and P329G mutation <400> 37 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 38 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a L234A, L235A,        P329G and hole mutation <400> 38 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 39 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 isotype with a L234A, L235A,        P329G and knob mutation <400> 39 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly 225 <210> 40 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 subclass with the mutations        L234A, L235A, P329G, Y349C, T366S, L368A and Y407V <400> 40 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro         115 120 125 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 41 <211> 221 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > variant human Fc-region of the IgGl subclass with a L234A, L235A,        P329G and S354C, T366W mutation <400> 41 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         115 120 125 Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 42 <211> 221 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > variant human Fc-region of the IgGl subclass with a L234A, L235A,        P329G and S354C, T366S, L368A and Y407V mutations <400> 42 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         115 120 125 Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 43 <211> 221 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > variant human Fc-region of the IgGl subclass with a L234A, L235A,        P329G and Y349C, T366W mutation <400> 43 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro         115 120 125 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 44 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 subclass with the mutations        I253A, H310A and H435A <400> 44 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         115 120 125 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         195 200 205 Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 45 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 subclass with the mutations        H310A, H433A and Y436A <400> 45 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         115 120 125 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu Ala         195 200 205 Asn His Ala Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 46 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG1 subclass with the mutations        M252Y, S254T and T256E <400> 46 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro             20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 85 90 95 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         115 120 125 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     210 215 220 <210> 47 <211> 228 <212> PRT <213> Homo sapiens <400> 47 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val     50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser             100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln     130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu             180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser         195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     210 215 220 Leu Ser Leu Gly 225 <210> 48 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG4 isotype with a S228P and        L235E mutation <400> 48 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val     50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser             100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln     130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu             180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser         195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     210 215 220 Leu Ser Leu Gly 225 <210> 49 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG4 isotype with a S228P, L235E        and P329G mutation <400> 49 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val     50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly Ser             100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln     130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu             180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser         195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     210 215 220 Leu Ser Leu Gly 225 <210> 50 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG4 isotype with a S228P and        L235E mutation <400> 50 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val     50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly Ser             100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln     130 135 140 Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu             180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser         195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     210 215 220 Leu Ser Leu Gly 225 <210> 51 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> variant human Fc-region of the IgG4 isotype with a S228P, L235E        and P329G mutation <400> 51 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val     50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly Ser             100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln     130 135 140 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu             180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser         195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     210 215 220 Leu Ser Leu Gly 225 <210> 52 <211> 107 <212> PRT <213> Homo sapiens <400> 52 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 53 <211> 105 <212> PRT <213> Homo sapiens <400> 53 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe             20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val         35 40 45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys     50 55 60 Tyr Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu                 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser             100 105 <210> 54 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 54 Gly Gly Gly Ser One <210> 55 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 55 Gly Gly Gly Gly Ser 1 5 <210> 56 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 56 Gln Gln Gln Ser One <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 57 Gln Gln Gln Gln Ser 1 5 <210> 58 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 58 Ser Ser Ser One <210> 59 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 59 Ser Ser Ser Ser 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 60 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 61 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 62 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 62 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 <210> 63 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 63 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser             20 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 64 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 65 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 65 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 66 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 66 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser             20 <210> 67 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 67 Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 1 5 10 15 Gly      <210> 68 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 68 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser          <210> 69 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 69 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly             20 <210> 70 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> peptidic linker <400> 70 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser             20 25 30 <210> 71 <211> 118 <212> PRT <213> Mus musculus <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr             20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Ile         35 40 45 Ala Met Ile Tyr Tyr Asp Ser Ser Lys Met Asn Tyr Ala Asp Thr Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Val Pro Thr Ser His Tyr Val Val Asp Val Trp Gly Gln Gly Val             100 105 110 Ser Val Thr Val Ser Ser         115 <210> 72 <211> 107 <212> PRT <213> Mus musculus <400> 72 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu 1 5 10 15 Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile         35 40 45 Tyr Gly Ala Thr Ser Leu Ala Asp Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Arg Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Arg Val Gln Val 65 70 75 80 Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Asn Thr Pro Trp                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys             100 105 <210> 73 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> anti-TfR mAb 8D3 VL variant L104V L106I <400> 73 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu 1 5 10 15 Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile         35 40 45 Tyr Gly Ala Thr Ser Leu Ala Asp Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Arg Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Arg Val Gln Val 65 70 75 80 Glu Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Asn Thr Pro Trp                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 74 <211> 122 <212> PRT <213> Oryctolagus cuniculus <400> 74 Gln Ser Met Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala             20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly         35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Gly     50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Arg Tyr                 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Asn Gly Phe Asp Pro Trp             100 105 110 Gly Pro Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 75 <211> 110 <212> PRT <213> Oryctolagus cuniculus <400> 75 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile         35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly     50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Cys Tyr Ser Ser Ser Asn                 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys             100 105 110 <210> 76 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 76 Gly Phe Ser Leu Ser Ser Tyr 1 5 <210> 77 <211> 5 <212> PRT <213> Oryctolagus cuniculus <400> 77 Trp Ser Gly Gly Ser 1 5 <210> 78 <211> 17 <212> PRT <213> Oryctolagus cuniculus <400> 78 Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Asn Gly Phe Asp 1 5 10 15 Pro      <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 299-000 HVR-H3 DASG <400> 79 Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Ser Gly Phe Asp 1 5 10 15 Pro      <210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 299-000 HVR-H3 DAQG <400> 80 Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Gln Gly Phe Asp 1 5 10 15 Pro      <210> 81 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 299-000 HVR-L1 RAA <400> 81 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala 1 5 10 <210> 82 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 82 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 83 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 299-000 HVR-L3 NYA <400> 83 Gln Gln Asn Tyr Ala Ser Ser Asn Val Asp Asn Thr 1 5 10 <210> 84 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Anti-TfR mAb 567 VH (= humanized 299 VH_basic) <400> 84 Gln Ser Met Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala             20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly         35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Gly     50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Arg Tyr                 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Asn Gly Phe Asp Pro Trp             100 105 110 Gly Pro Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 85 <211> 110 <212> PRT <213> Artificial Sequence <220> Anti-TfR mAb 567 VL (= humanized 299 VL_basic) <400> 85 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile         35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly     50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Ser 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Ser Ser Ser Asn                 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys             100 105 110 <210> 86 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> anti-TfR mAb 932 VH (= humanized 299 VH_mutated) <400> 86 Gln Ser Met Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala             20 25 30 Met Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly         35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Ser     50 55 60 Arg Val Thr Ile Ser Lys Thr Ser Thr Thr Val Ser Leu Lys Leu Ser 65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Tyr                 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Asn Gly Phe Asp Pro Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 87 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> anti-TfR mAb 932 VL (= humanized 299 VL_mutated) <400> 87 Ala Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr Ala Ser Ser Asn                 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 88 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> 299-023 VH humanization variant_DASG <400> 88 Gln Ser Met Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala             20 25 30 Met Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly         35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Ser     50 55 60 Arg Val Thr Ile Ser Lys Thr Ser Thr Thr Val Ser Leu Lys Leu Ser 65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Tyr                 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Ser Gly Phe Asp Pro Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 89 <211> 119 <212> PRT <213> Mus musculus <400> 89 Glu Val Gln Leu Gln Gln Ser Gly Ala Val Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Pro Ala Ser Gly Phe Asn Ile Lys Asp Thr             20 25 30 Tyr Ile His Trp Val Ile Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile         35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys Cys Asp Pro Lys Phe     50 55 60 Gln Val Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys                 85 90 95 Val Arg Asp Tyr Leu Tyr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln Gly             100 105 110 Thr Thr Leu Thr Val Ser Ser         115 <210> 90 <211> 108 <212> PRT <213> Mus musculus <400> 90 Lys Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Ser Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Asn Cys Arg Ala Ser Glu Ser Val Asp Thr Tyr             20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Glu Leu Leu Ile         35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr Tyr Cys Gly Gln Thr Tyr Asn Tyr Pro Leu                 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg             100 105 <210> 91 <211> 118 <212> PRT <213> Mus musculus <400> 91 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr             20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Asn Pro His Asn Gly Gly Thr Asp Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Tyr Tyr Tyr Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 92 <211> 106 <212> PRT <213> Mus musculus <400> 92 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Arg Tyr Ile             20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr         35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Ser Ser Phe Ser Gly Ser     50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Asn Ser Tyr Pro Trp Thr                 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 93 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> human transferrin receptor fragment <400> 93 Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser Asn Gly Asn Leu 1 5 10 15 <210> 94 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> human transferrin receptor fragment <400> 94 Asn Met Val Thr Ile Val Gln Ser Asn Gly Asn Leu Asp Pro Val 1 5 10 15 <210> 95 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> human transferrin receptor fragment <400> 95 Gln Ser Asn Gly Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr 1 5 10 15 <210> 96 <211> 693 <212> PRT <213> Artificial Sequence <220> <223> anti-Her2 circular fusion polypeptide <400> 96 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr             20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val         115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly     210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 225 230 235 240 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe                 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val             260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe         275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro     290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val                 325 330 335 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala             340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg         355 360 365 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly     370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser                 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln             420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His         435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly     450 455 460 Ser Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 465 470 475 480 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser                 485 490 495 Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys             500 505 510 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val         515 520 525 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr     530 535 540 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 545 550 555 560 His Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile                 565 570 575 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp             580 585 590 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn         595 600 605 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu     610 615 620 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 625 630 635 640 Ser Thr Ser Ser Ser Ser Thr Ser Ser Ser Ser Thr Leu                 645 650 655 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser             660 665 670 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Ser Gly His         675 680 685 His His His His His     690 <210> 97 <211> 692 <212> PRT <213> Artificial Sequence <220> <223> anti-cMet circular fusion polypeptide <400> 97 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser     210 215 220 Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro                 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val             260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala                 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro             340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr         355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser     370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr                 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe             420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys         435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly     450 455 460 Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 465 470 475 480 Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr                 485 490 495 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu             500 505 510 Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Leu Pro         515 520 525 Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln     530 535 540 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 545 550 555 560 Tyr Cys Ala Pro Ser Tyr Tyr Tyr Gly Gly Lys His Val Ala Leu Phe                 565 570 575 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr             580 585 590 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser         595 600 605 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu     610 615 620 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 625 630 635 640 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser                 645 650 655 Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys             660 665 670 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu         675 680 685 Pro Lys Ser Cys     690 <210> 98 <211> 693 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 circular fusion polypeptide (1) <400> 98 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile         35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly             100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu                 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu             260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys         275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys     290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys             340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln             420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly     450 455 460 Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln Ser Pro Ala 465 470 475 480 Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala                 485 490 495 Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser             500 505 510 Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val         515 520 525 Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr     530 535 540 Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 545 550 555 560 Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile                 565 570 575 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp             580 585 590 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn         595 600 605 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu     610 615 620 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 625 630 635 640 Ser Thr Ser Ser Ser Ser Thr Ser Ser Ser Ser Thr Leu                 645 650 655 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser             660 665 670 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Ser Gly His         675 680 685 His His His His His     690 <210> 99 <211> 697 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Anti-CD20 circular fusion polypeptide (2) <400> 99 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser             20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe         115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu     130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu                 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser             180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro         195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly     210 215 220 Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             260 265 270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     290 295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         355 360 365 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 405 410 415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser     450 455 460 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu 465 470 475 480 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys                 485 490 495 Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln             500 505 510 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu         515 520 525 Val Ser Gly Val Ser As Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser     530 535 540 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 545 550 555 560 Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr                 565 570 575 Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe             580 585 590 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys         595 600 605 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val     610 615 620 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 625 630 635 640 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser                 645 650 655 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His             660 665 670 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys         675 680 685 Gly Ser Gly His His His His His His     690 695 <210> 100 <211> 689 <212> PRT <213> Artificial Sequence <220> <223> anti-cMet circular fusion polypeptide VH-out-knob <400> 100 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp             20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp         35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly     50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Pro Ser Tyr Tyr Tyr Gly Gly Lys His Val Ala Leu Phe Ala Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly     130 135 140 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val         195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys     210 215 220 Ser Cys Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 465 470 475 480 Gln Ser Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile                 485 490 495 Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln             500 505 510 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val Tyr Asn Ala Lys Thr         515 520 525 Leu Ala Glu Gly Val Ser Ser Gly Ser Gly Ser     530 535 540 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 545 550 555 560 Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe Thr Phe Gly Gln Gly                 565 570 575 Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile             580 585 590 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val         595 600 605 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys     610 615 620 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 625 630 635 640 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu                 645 650 655 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr             660 665 670 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu         675 680 685 Cys      <210> 101 <211> 692 <212> PRT <213> Artificial Sequence <220> <223> anti-cMet circular fusion polypeptide VH-in-knob <400> 101 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser     210 215 220 Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro                 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val             260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala                 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro             340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr         355 360 365 Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser     370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr                 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe             420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys         435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly     450 455 460 Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 465 470 475 480 Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr                 485 490 495 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu             500 505 510 Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Leu Pro         515 520 525 Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln     530 535 540 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 545 550 555 560 Tyr Cys Ala Pro Ser Tyr Tyr Tyr Gly Gly Lys His Val Ala Leu Phe                 565 570 575 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr             580 585 590 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser         595 600 605 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu     610 615 620 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 625 630 635 640 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser                 645 650 655 Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys             660 665 670 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu         675 680 685 Pro Lys Ser Cys     690 <210> 102 <211> 685 <212> PRT <213> Artificial Sequence <220> <223> anti-cMet circular fusion polypeptide VH-out-hole <400> 102 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ala Tyr             20 25 30 Phe Ile Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Tyr Pro Tyr Asn Gly Asn Thr Phe Tyr Asp Gln Ser Phe     50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Trp Asp Tyr Asn Tyr Asp Val Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala         115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu     130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser                 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu             180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr         195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser     210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu                 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu             260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys         275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys     290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys             340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser         355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys     370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 405 410 415 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln             420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly     450 455 460 Gly Ser Gly Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala 465 470 475 480 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala                 485 490 495 Ser Ser Ser Val Ser His Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln             500 505 510 Ala Pro Arg Leu Trp Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val         515 520 525 Pro Ala Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr     530 535 540 Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys His Gln 545 550 555 560 Arg Thr Asn Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile                 565 570 575 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp             580 585 590 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn         595 600 605 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu     610 615 620 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 625 630 635 640 Ser Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Thr                 645 650 655 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu             660 665 670 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys         675 680 685 <210> 103 <211> 686 <212> PRT <213> Artificial Sequence <220> <223> anti-cMet circular fusion polypeptide VH-out-hole-CH-CL-crossed <400> 103 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ala Tyr             20 25 30 Phe Ile Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Tyr Pro Tyr Asn Gly Asn Thr Phe Tyr Asp Gln Ser Phe     50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Trp Asp Tyr Asn Tyr Asp Val Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser Ala Val Ala Ala Pro Ser Val Phe Ile Phe Pro         115 120 125 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu     130 135 140 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 145 150 155 160 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp                 165 170 175 Ser Lys Asp Ser Thr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys             180 185 190 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln         195 200 205 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly     210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr         355 360 365 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser     370 375 380 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr 465 470 475 480 Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu                 485 490 495 Ser Cys Arg Ala Ser Ser Ser Val Ser His Leu Tyr Trp Tyr Gln Gln             500 505 510 Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr Asp Thr Ser Asn Leu         515 520 525 Ala Ser Gly Val Ala Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp     530 535 540 Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr 545 550 555 560 Phe Cys His Gln Arg Thr Asn Tyr Pro Trp Thr Phe Gly Gln Gly Thr                 565 570 575 Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe             580 585 590 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu         595 600 605 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp     610 615 620 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 625 630 635 640 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser                 645 650 655 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro             660 665 670 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys         675 680 685 <210> 104 <211> 683 <212> PRT <213> Artificial Sequence <220> <223> anti-cMet circular fusion polypeptide VH-out-hole-VH-VL-crossed <400> 104 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser His Leu             20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr         35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Ala Arg Phe Ser Gly Ser     50 55 60 Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Phe Cys His Gln Arg Thr Asn Tyr Pro Trp Thr                 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys             100 105 110 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly         115 120 125 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro     130 135 140 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 145 150 155 160 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val                 165 170 175 Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn             180 185 190 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro         195 200 205 Lys Ser Cys Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp     210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu             260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg     290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu                 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys             340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu         355 360 365 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp                 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His             420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln Leu     450 455 460 Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile 465 470 475 480 Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ala Tyr Phe Ile Asn Trp                 485 490 495 Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Arg Ile Tyr             500 505 510 Pro Tyr Asn Gly Asn Thr Phe Tyr Asp Gln Ser Phe Gln Gly Gln Val         515 520 525 Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser     530 535 540 Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Trp Asp 545 550 555 560 Tyr Asn Tyr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser                 565 570 575 Ser Ala Val Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu             580 585 590 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe         595 600 605 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln     610 615 620 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 625 630 635 640 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu                 645 650 655 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser             660 665 670 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys         675 680 <210> 105 <211> 120 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE &Lt; 222 > (131) .. (131) <223> X = E or D <220> <221> MISC_FEATURE <133> (133) <223> X = M or L <400> 105 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys         115 120

Claims (14)

As dimeric fusion polypeptides,
A first fusion polypeptide that specifically binds to a target comprising a first portion of a binding domain, a second portion of a binding domain, and a spacer domain, such as:
The spacer domain is a polypeptide and comprises at least 25 amino acid residues,
The first portion of the binding domain is a polypeptide fused directly or through the first linker to the N-terminus of the spacer domain,
The second portion of the binding domain is a polypeptide fused directly or through the second linker to the C-terminus of the spacer domain,
The first portion of the binding domain of the same short chain fusion polypeptide and the second portion of the binding domain are associated to form a functional binding site that is specifically bound to the target,
And
A second fusion polypeptide that specifically binds to a target comprising a first portion of a binding domain, a second portion of a binding domain, and a spacer domain, such as:
The spacer domain is a polypeptide and comprises at least 25 amino acid residues,
The first portion of the binding domain is a polypeptide fused directly or through the first linker to the N-terminus of the spacer domain,
The second portion of the binding domain is a polypeptide fused directly or through the second linker to the C-terminus of the spacer domain,
The first portion of the binding domain of the same short chain fusion polypeptide and the second portion of the binding domain are associated to form a functional binding site that specifically binds to the target,
Lt; / RTI &gt;
Wherein the first and second fusion polypeptides are the same or different and the spacer domain of the first fusion polypeptide is covalently conjugated to the spacer domain of the second fusion polypeptide. 2. The dimer fusion polypeptide of claim 1, wherein the first portion of the binding domain is an antibody heavy chain variable domain and the second portion of the binding domain is an antibody light chain variable domain or vice versa. 3. The dimer fusion polypeptide of claim 1 or 2, wherein the first portion of the binding domain is an antibody heavy chain Fab fragment and the second portion of the binding domain is an antibody light chain Fab fragment or vice versa. 7. The dimer fusion polypeptide of claim 1, wherein the fusion polypeptide is free of an antibody variable domain. 5. A dimer fusion polypeptide according to any one of claims 1 to 4, wherein the first portion of the binding domain and the second portion of the binding domain are covalently associated with each other by a disulfide bond. 6. A dimer fusion polypeptide according to any one of claims 1 to 5, wherein the spacer domain comprises an antibody hinge region or a (C-terminal) fragment thereof and an antibody CH2 domain or an (N-terminal) fragment thereof. 7. A dimer fusion polypeptide according to any one of claims 1 to 6, wherein the spacer domain comprises an antibody hinge region or fragment thereof, an antibody CH2 domain, and an antibody CH3 domain or fragment thereof. 8. A dimer fusion polypeptide according to any one of claims 1 to 7, wherein the first and / or second linker is a peptide linker. A pair of isolated nucleic acids that together encode the dimeric fusion polypeptide of claim 1. 12. A host cell comprising an isolated nucleic acid pair according to claim 9. A method of producing a fusion polypeptide comprising culturing the host cell of claim 10 to produce a fusion polypeptide or a dimeric fusion polypeptide and recovering the fusion polypeptide or dimer fusion polypeptide from the cell or culture medium. A pharmaceutical formulation comprising a dimeric fusion polypeptide according to claim 1 and a pharmaceutically acceptable carrier. The dimer fusion polypeptide of claim 1, for use as a medicament. Use of a dimeric fusion polypeptide according to claim 1 in the manufacture of a medicament.

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